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Monitoring the Effects of Synthetic Lung Surfactant Peptides on Lipid Bilayer Dynamics and Fluidity

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Title:
Monitoring the Effects of Synthetic Lung Surfactant Peptides on Lipid Bilayer Dynamics and Fluidity
Creator:
Braide, Otonye H
Place of Publication:
[Gainesville, Fla.]
Florida
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University of Florida
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Language:
english
Physical Description:
1 online resource (146 p.)

Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Chemistry
Committee Chair:
FANUCCI,GAIL E
Committee Co-Chair:
TALHAM,DANIEL R
Committee Members:
OMENETTO,NICOLO
SMITH,BEN W
LONG,JOANNA R
Graduation Date:
5/3/2014

Subjects

Subjects / Keywords:
Anisotropy ( jstor )
Chemicals ( jstor )
Fluorescence ( jstor )
Lipids ( jstor )
Liposomes ( jstor )
Lungs ( jstor )
pH ( jstor )
Phospholipids ( jstor )
Surfactants ( jstor )
Wavelengths ( jstor )
Chemistry -- Dissertations, Academic -- UF
biomembrane -- hypophase -- kl4 -- lipids -- liposomes -- lung -- phospholipids -- pulmonary -- respiratory -- sp-b -- surfactant
Genre:
bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
born-digital ( sobekcm )
Electronic Thesis or Dissertation
Chemistry thesis, Ph.D.

Notes

Abstract:
Premature infants with underdeveloped lungs typically develop respiratory distress syndrome (RDS) because their lungs lack the surfactant lining crucial for oxygen absorption or have genetically failed to produce critical components of lung surfactant (LS) needed for proper function. The LS is a complex mixture of lipids and proteins known to provide a protective barrier against inhaled pathogens, lower alveolar surface tension and promote oxygen exchange. The functional significance of LS is clear; however, a detailed mechanistic understanding of how lipids are trafficked to and from the air-fluid interface for oxygen absorption remains unknown. Though the bulk of LS is made up of lipids (~90%), it is non-functional without the presence of surfactant proteins (SP-A, B, C, and D), especially SP-B, which is known to reduce surface tension and closely associates with lipids in the bulk phase of liquid below alveoli surface film (hypophase). As SP-B is highly hydrophobic and structurally complex, challenges in synthesis and expression of a functionally active recombinant have led to increased efforts to use synthetic alternatives in developing novel therapeutics for RDS treatment, and to elucidate the mechanism of function. Within the scope of this dissertation, we use alternatives such as KL4, a mimetic of SP-B, and SP-B59-80; which have been shown to retain the fundamental properties necessary for LS function when in the presence of major phospholipid constituents, as model systems to further our understanding of fundamental membrane-protein interactions and of specific LS components function. Fluorescence and electron paramagnetic resonance techniques were used to investigate the effects of the synthetic peptides on lipid dynamics, fluidity, and to model peptide depth profiles in lipid bilayers. The results from these experiments suggested a deeper penetration of the peptide into lipid systems that are rich in the main lipid found at the air-fluid interface monolayer; and also showed induction of curvature strains in the bilayer, both of which correlate with the hypothesized mechanism of lipid flipping to the air-fluid interface. Circular dichroism was used to evaluate pH effects on peptide conformation, which highlighted the importance of using biologically relevant conditions to accurately model the molecular biophysics of LS. ( en )
General Note:
In the series University of Florida Digital Collections.
General Note:
Includes vita.
Bibliography:
Includes bibliographical references.
Source of Description:
Description based on online resource; title from PDF title page.
Source of Description:
This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis:
Thesis (Ph.D.)--University of Florida, 2014.
Local:
Adviser: FANUCCI,GAIL E.
Local:
Co-adviser: TALHAM,DANIEL R.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2014-11-30
Statement of Responsibility:
by Otonye H Braide.

Record Information

Source Institution:
UFRGP
Rights Management:
Applicable rights reserved.
Embargo Date:
11/30/2014
Classification:
LD1780 2014 ( lcc )

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MONITORING THE EFFECTS OF SYNTHETIC LUNG SURFACTANT PEPTIDES ON LIPID BILAYER DYNAMICS AND FLUIDITY By OTONYE BRAIDE A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF T HE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2014

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2014 Otonye Braide

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To God be the glory

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4 ACKNOWLEDGMENTS First I would like to thank God for His mercy, grace, love and faithfuln ess; and for His endless provision physically and spiritually. I would also like to thank my advisor, Dr Gail Fanucci, for her ability to challenge and stimulate intellectual growth, numerous educational and personal discussions, and for her consistent a nd unfailing support through major milestones these past few y ears. I am also grateful to her for giving me opportunities to attend multiple conferences and for inspiring a new interest in biomembranes through her special topics course. With equal regard, I would like to thank my co advisor, Dr Joanna Long, for her infectious passion for research, for exuding positivity no matter the circumstance, and for being an example of dilig ence. Through their supervision I was able to experience true mentorship an d benefited from a multitude of insightful feedback and discussions I would like to thank Dr Ben Smith for believing in me and for his support, especially through hard transi tions within my graduate career, and for leading me to a research group where I could develop as a scientist. A special thanks is extended to Dr Nicolo Omenetto for his enthusiasm, and helpful discussions on spectroscopy and experimental design. Along with Dr Daniel Talham, I am grateful for their willingness to serve on my disser tation committee. I would like to thank Dr Ann e Do nnelly for continual mentorship and friendship, mental and financial supp or t. For being by my side throughout my graduate career at UF and for never wavering in her perspective of what could be achieved For her keen desire to see all students succeed whether they were members of the South East Alliance for Graduate Education and the Professoriate (SEAGEP) program or not. For giving m e the opportunity to serve as a Research Experience for Undergraduates (REU)

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5 coordinator for two years, and for sending me to the C ompact for Faculty Diversity Institute on Teaching and Mentoring conference twice; both of which were inspiring Words truly cannot express the depths of what her mentorship and friendship ha s mea nt, even from a distance. A special thanks to Dr. Hagen for generously allowing me to use his lab space and circular dichroism (CD) instrument. Through the years, many previous and current labmates, were a source of support and friendship: Dr Kane Barke r, Dr Lindsay Sexton, Dr Jillian Perry, Dr Kaan Kececi, Funda Tongay, William Hardy, Dr Pu Jin, Dr Stacey Ann Benjamin, Dr Anna Kuznetsova, Dr. Xi Huang, Dr Ian De Vera, Adam Smith and all other members of the Long and Fanucci research groups. Spec ial recognition is extended to: Dr Hitomi Mukaibo for her continuous support and advice through the years; Dr Suzanne Farver for her patience in training me and sincerity; Dr Yong Ran for his training and willingness to impart his knowledge and expertis e on various subject matters ; Katye Poole for her candor and willingness to help at all times, truly a key player in some of the major milestones. I am grateful for a labmate who developed into a cherished friend, a sister who g enuinely ex pressed love wi th humbling truth when needed and always extended a listening ear through the final stretch of this process To Ms. Vivian Thompson for her kindness, patience, and encouragement through the years; Ms. Lori Clark for tolerating my absent mindedness a multitude of times and her supp ort; and Ms. Glennis Brown for allowing me to camp out in her office and the many life based conversations.

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6 I would like to thank members of the Board of Education (BOE) program, especially Ms. Sarah Pe rry for her consistent willingness to help; and to Mr. Earl Wade for his support. To Dr. Henry Frierson, De an of the Graduate School at UF; D r. Lawrence Morehouse, Mr. Charles Jackson Ms. Phyllis Reddick, Ms. Lyra Logan and other McKnigh t Fellows program members ; for their positivity, willingness to listen, and support To Dr. Kachina Allen for her patience, sincerit y and generosity in helping me to communicate more clearly. To Dr Goro Nagase and the late Professor Lin, for challenging me intellectually b eyond the general curriculum in mathematics and chemistry, respectively. And a special thanks to Dr Geneive Henry for making organic che mistry one of the best class es to date. I would like to thank my parents, Mr Sonny Oko Braide and Mrs Queenba Braide for raising me as their own, and all their support throughout the years. I would like to thank my mom, Mrs Joan Oehlert for c hoosing to reach out and connect with me, for taking an active interest in my life, being a friend and for her overwhelming generosity, especially during my graduate career. I am fortunate to have her in my life. To Dr. Johnny R Johnson for his unwavering support and for taking a genuine interest in my life since I was a teenager

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7 I would like to thank Ms Linda Brown for her love, acceptance, relentless affirmation and belief in my potential for the past 12+ years of my life A special thanks is extended to Pastor Glenn and Mrs Valerie Vellekamp for their counsel, unconditional love and encouragement through hard times. F or choosing to adopt me into their family without condition and f or the many nights I found shelter and comfort in their home To the fr iends and family I found within the Greenhouse Church, formerly known as First Assembly of God, and those out of the church who truly made Gainesville home to me through the years o ruly had no one to turn to and for consistently being an example of truth and love. To my little sister, Destiny Ayanna Hampton, for making the past few years rich with love and true tests of character. To Ronald Moncoeur for his relentless support duri ng the last wave of challenges and for his companionship.

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8 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ .......... 11 LIST OF FIGURES ................................ ................................ ................................ ........ 12 LIST OF ABBREVIATIONS ................................ ................................ ........................... 16 ABSTRACT ................................ ................................ ................................ ................... 20 CHAPTER 1 INTRODUCTION AND RESEARCH OVERVIEW ................................ ................... 22 Lung Surfactant ................................ ................................ ................................ ...... 22 Life Cycle and Function ................................ ................................ .................... 24 Composition ................................ ................................ ................................ ..... 25 Lipid Structures and Functions ................................ ................................ ... 26 Protein Structures and Functions ................................ ............................... 30 Respiratory Conditions and T reatments ................................ ................................ .. 31 Research Overview ................................ ................................ ................................ 33 2 INSTRUMENTATION THEORY AND METHODS USED ................................ ....... 42 Introduction ................................ ................................ ................................ ............. 42 Thin Layer Chromatography ................................ ................................ ................... 42 Differential Scanning Calorimetry ................................ ................................ ........... 44 Spectroscopy ................................ ................................ ................................ .......... 45 Light ................................ ................................ ................................ ................. 45 Matter ................................ ................................ ................................ ............... 47 UV Vi s Spectroscopy ................................ ................................ .............................. 48 Spectrophotometer ................................ ................................ ........................... 49 Signal Output ................................ ................................ ................................ .... 50 Circular Di chroism (CD) Spectroscopy ................................ ................................ ... 51 Polarized Light ................................ ................................ ................................ .. 52 Application ................................ ................................ ................................ ........ 52 Fluo rescence Spectroscopy ................................ ................................ .................... 53 Fluorometer ................................ ................................ ................................ ...... 54 Signal Output ................................ ................................ ................................ .... 55 Advantages and Disadvantages ................................ ................................ ....... 57 Electron Paramagnetic Resonance ................................ ................................ ........ 58 Timescales of Lipid Motions ................................ ................................ .................... 59

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9 3 EXPERIMENTAL OPTIMIZATION ................................ ................................ .......... 72 Introduction ................................ ................................ ................................ ............. 72 Vesicle Preparation ................................ ................................ ................................ 72 Lipid Structure and Concentration ................................ ................................ .... 73 Liposome Formation ................................ ................................ ......................... 74 Fluorescent Probes ................................ ................................ ................................ 76 Pyrene ................................ ................................ ................................ .............. 76 DPH and TMA DPH ................................ ................................ ......................... 78 Rhodamine PE ................................ ................................ ................................ 79 Experimental Parameters and Results ................................ ................................ .... 80 PPDPC ................................ ................................ ................................ ............. 80 DPH ................................ ................................ ................................ .................. 82 TMA DPH ................................ ................................ ................................ ......... 83 Rhodamine PE ................................ ................................ ................................ 84 Discussion and Conclusions ................................ ................................ ................... 84 4 EF FECTS OF pH ON KL 4 PEPTIDE STRUCTURE AT MEMBRANE INTERFACE ................................ ................................ ................................ ........... 93 Introduction ................................ ................................ ................................ ............. 93 Surface Potential ................................ ................................ .............................. 93 Surface pH ................................ ................................ ................................ ....... 95 Material and Methods ................................ ................................ ............................. 97 Synthesis of KL 4 ................................ ................................ ............................... 97 Preparation of Liposome Samples ................................ ................................ ... 97 CD Experiments ................................ ................................ ............................... 98 Results and Discussion ................................ ................................ ........................... 98 Conclusions ................................ ................................ ................................ .......... 100 5 PEPTIDE EFFECTS ON MEMBRANE ORGANIZATION AND DYNAMICS ......... 106 Abstract ................................ ................................ ................................ ................. 106 Introduction ................................ ................................ ................................ ........... 107 Material and Methods ................................ ................................ ........................... 109 Synthesis of KL 4 ................................ ................................ ............................. 109 Preparation of Liposome Samples ................................ ................................ 110 Measurement of I e /I m for Samples Containing PPDPC ................................ ... 110 Fluorescence Anisotropy of Samples containing Rhodamine PE ................... 111 Results and Discussion ................................ ................................ ......................... 111 Selective KL 4 Partitioning into DPPC:POPG bilayers ................................ ..... 111 Collisional Quenching of Headgroup Tethered Probe ................................ .... 112 Conclusions ................................ ................................ ................................ .......... 114 6 ACCESSIBILITY STUDIES WITH SPB C AND SPIN LABELED LIPIDS ............. 119 Introduction ................................ ................................ ................................ ........... 119

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10 Material and Method s ................................ ................................ ........................... 119 Synthesis of SP B 59 80 ................................ ................................ .................... 119 Buffer Solutions Protocol ................................ ................................ ................ 120 Prep aration of Liposome Samples for Nickel Determination .......................... 120 Preparation of Liposomes containing Surfactant Peptide ............................... 121 CW EPR Spectra Acquisition ................................ ................................ ......... 121 Power Saturation Experiments ................................ ................................ ....... 122 Results and Discussion ................................ ................................ ......................... 123 Nickel Determination Experiments ................................ ................................ 123 Accessibility Measurements with SP B 59 80 Assimilation ................................ 124 Conclusions ................................ ................................ ................................ .......... 125 7 CONCLUSIONS AND FUTURE DIRECTIONS ................................ .................... 132 LIST OF REFERENCES ................................ ................................ ............................. 134 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 146

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11 LIST OF TABLES Table page 1 1. Lipid composition of extracellular surfactant in selected mammalian species by weight. (Adapted fr om The role of lipids in lung surfactant by Ruud Veldhuizen et al 7 ) ................................ ................................ ............................... 36 2 1. Molecular moieties likely to absorb light in the UV Vis region ............................ 64 6 1. Standard CW EPR parameters used for power saturation experiments ........... 127

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12 LIST OF FIGURES Figure page 1 1. Esti mated rates of preterm births in 2010 (adopted from World Health Born Too Soon: The Global Action Report on Preterm Birth ). 124 ................................ ................................ ................................ .............. 34 1 2. Lung surfactant life cycle (http:/ /www.jped.com.br/conteudo/01 77 S3/ing_print.htm#1). ................................ ................................ ........................... 35 1 3. Chemical structures of main glycerophospholipids found in surfactant: A) DPPC B) POPC and C) POPG. ................................ ................................ .......... 36 1 4. General classification of eukaryotic lipids (modified from Nelson and Cox). 61 .... 37 1 5. Chemical structures of A) glycerol B) fatt y acid C) an example of an acyl chain (blue) and D) glycerophospholipid where R represents an acyl chain of 3 or more carbons and X is a headgroup (choline, glycerol, etc.). ...................... 38 1 6. Li pid polymorphisms: fluid (L ), gel (L ), hexagonal (H I ), inverted hexagonal (H II ) and various isotropic phases. 62 ................................ ................................ ... 38 1 7. Morphology of different sizes/types of liposomes in 2 dimensions. .................... 39 1 8. Surfactant proteins A, C and D models were generated in MacPymol (Schrdinger, LLC); SP B was adopted from Olmeda et al. 63 ............................. 40 1 9. Sequence alignments for SP B, SP B 1 25 SP B 59 80 and synthetic peptide mimic KL 4 showing charged residues highlighted; basic in blue and acidic in red ................................ ................................ ................................ ..................... 41 2 1. Thin layer chromatog ram of the negative control LPC, DPPC:POPG and POPC:POPG mixtures, visualized by staining with iodine vapour. Each phospholipid component is circled ................................ ................................ ...... 61 2 2. Electromagnetic spectrum. ................................ ................................ ................. 62 2 3. Plane polarized electromagnetic radiation propagating along x axis (http://micro.magnet.fsu.edu/primer/java/wavebasics/). ................................ ...... 62 2 4. Molecular energy level diagram showing the transition from the ground state (S 0 ) to first (S 1 ) level excited state, internal conversion, vibrational relaxation and fluorescence: ................................ ................................ .............................. 63 2 5. Correlation diagram of the energy states of hydrogen atoms before and after formation of diatomic h ydrogen (H 2 ): lowest energy state is the bonding ....... 63

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13 2 6. Electronic transitions and respective energy leve ls. ................................ ........... 64 2 7. UV Vis Spectrophotometer schematic. ................................ ............................... 65 2 8. Absorption spectrum of cuvettes made from different mate rials (containing water) and possible wavelengths of interference. ................................ ............... 65 2 9. The formation of elliptically polarized light from left (E L ) and right (E R ) circularly polarized light and th ................................ ......... 66 2 10. Graphical representation of polarized light shifted out of phase to produce circularly polarized light. ................................ ................................ ..................... 67 2 11. Peptide bond transitions. ................................ ................................ .................... 67 2 12. Circular dichroism standard protein structure spectra (http://www.proteinchemist.com/cd/cdspec.html). ................................ ............... 68 2 13. Absorption and fluorescence emission spectra. ................................ ................. 68 2 14. Block diagram showing general schematic of spectrofluorometer. ..................... 69 2 15. A schematic of the spectrofluorometer components, as displayed in the Fluoromax 3 manual. 1 Xenon arc l amp and lamp housi ng, 1a Xenon lamp power supply, ................................ ................................ ................................ .... 69 2 16. Energy level diagram for a free electron in an applied magnetic field showing the Zeeman int eraction. ................................ ................................ ...................... 70 2 17. Energy diagram showing the hyperfine splitting of an electron with m s = 1/2 coupled to a nucleus with m I = 1. ................................ ................................ ........ 70 2 18. EPR spectral line shapes for labeled at the headgroup (tempo) and acyl chain (5 7 and 12 doxyl) regions of a lipid. ................................ ..................... 71 2 19. Timescales of different types of lipid motions in a lipid bilayer model (adopted from Adam Smith). ................................ ................................ .............................. 71 3 1. Fluorescent probes used throughout this dissertation. ................................ ....... 86 3 2. A) Malachite green phosphate assay standard curve used to determine phospholipid concentration prior to sample preparation: the error bars represent the deviation ................................ ................................ ...................... 86 3 3. Mini extruder set up with holder/heating block and mounted syringes (Avanti Polar Lipids, Alabaster, AL). ................................ ................................ ............... 87

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14 3 4 Emission spectra show concentration dependence effects on the excimer (I e ) and monomer (I m ) emissions of varied mol% PPDPC in A) 4:1 POPC:POPG and B) 4:1 DPPC:POPG liposomes. ................................ ................................ .. 88 3 5. Normalized fluorescence emission intensity of 1 to 10 mol% PPDPC in 4:1 DPPC:POPG (red circles) and 4:1 POPC:POPG (black triangles). A) The results of monomer em ission intensity ................................ ................................ 89 3 6. Thermotropic phase transitions of liposomes containing 3 mol% PPDPC. A) Results show the e xcimer to monomer (I e /I m ) values in 4:1 DPPC:POPG (red circles) and 4:1 POPC:POPG (black triangles) ................................ .................. 89 3 7. Fluorescence anisotropy results o f 4:1 DPPC:POPG (red circles) and 4:1 POPC:POPG (black triangles) with A) increasing DPH concentration: 0.01 to 5 mol%. ................................ ................................ ................................ ............. 90 3 8. Fluorescence anisotropy results of 4:1 DPPC:POPG (red circles) and 4:1 POPC:POPG (black triangles) with increasin g TMA DPH concentration: 1 to 10 mol%. ................................ ................................ ................................ ........... 91 3 9. Thermotropic phase transitions of liposomes containing 2 mol% TMA DPH. A) Results obtained from fluorescence anisotropy measurements 4:1 DPPC:POPG (red circles) ................................ ................................ ................. 91 3 10. Fluorescence anisotropy results of 4:1 DPPC:POPG (red circles) and 4 :1 POPC:POPG (black triangles) with A) increasing rhodamine PE concentration: 0.01 to 0.4 mol%. ................................ ................................ ........ 92 4 1. CD spectra of KL 4 in A) 4:1 DPPC:POPG and B) 4:1 POPC:POPG liposomes at 45 C Total lipid concentrations were 2 mM with 2 mol% peptide in a 10 mM NaH 2 PO 4 H 2 O/Na 2 HPO 4 ................................ ................................ .......... 103 4 2. CD spectra of KL 4 in A) 4:1 DPPC:POPG and B) 4:1 POPC:POPG liposomes at 45 C Total lipid concentrations were 2 mM with 2 mol% peptide in a 10 mM Bis Tris propane buffer ................................ ................................ .............. 104 4 3. Molar ellipticity ( ) at 222 nm as a function of pH values in 10 mM A) phosphate buffer and B) Bis Tris buffer solutions with 140 mM sodium chloride at pH 6.4, 6.7, 7.0, 7.3, 7.6 and 7.9. ................................ ................... 105 5 1. Effects of KL 4 concentration on rate of excimer formation as shown with the percent change in excimer to monomer ratios (I e /I m ) in 4:1 DPPC:POPG (hashed bars) and 4:1 POPC:POPG (solid bars) ................................ ............ 115 5 2. Hypothetical placement of PPDPC chemical structures on models 117 for the interaction of KL 4 with A) 3:1 POPC:POPG and B) 4:1 DPPC:POPG liposomes. Lipid coordinates were provided by Scott Fellers. 123 ...................... 116

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15 5 3. Effects of KL 4 concentration on relative changes in anisotropy (r) of 4:1 DPPC:POPG (hashed bars) and 4:1 POPC:POPG (solid bars) liposomes containing 0.05 mol% rhodamine PE. ................................ ............................. 117 5 4. Effects of KL 4 concentration on anisotropy (black squares) and fluorescence emissions (blue triangles) in 4:1 POPC:POPG liposomes containing 0.05 mol% rhodamine PE. ................................ ................................ ....................... 118 6 1. A) Chemical structure of nickel (II) acetylacetonate (NiAA) used to make buffers for power saturation expe riments. B) UV spectra of 0, 10, 20 and 30 mM NiAA buffer with 50 mM Bis Tris propane ................................ ................. 126 6 2 Standard curve of 10, 20 and 30 mM NiAA buffers made with 50 mM Bis Tris propane + 140 mM NaCl buffer at pH 6.5. ................................ ........................ 126 6 3. CW EPR spectrum of a standard nitroxide spin label showing th ree transition peaks. The peak to peak ( A pp ) amplitude is measured and used for power saturation curve plots. ................................ ................................ ...................... 127 6 4. Depth parameter plotted as a function of the following spin labeled lipids: tempo POPC, 5 7 and 12 doxyl PSPC (1 mol%). 10, 20 and 30 mM NiAA buffers made with 50 mM Bis Tris propane ................................ ..................... 128 6 5. Power saturation a ccessibility parameter P 1/2 (NiAA) plotted as a function of mol% SP B 59 80 in A) 4:1 DPPC:POPG and B) 4:1 POPC:POPG liposomes. Each contained 1 mol% ................................ ................................ ................... 128 6 6. 1/2 (O 2 ) plotted as a function of mol% SP B 59 80 in 4:1 DPPC:POPG and 4:1 POPC:POPG liposomes, which are separated according ................................ ................................ .................. 129 6 7. Power saturation accessibility parameter P 1/2 (NiAA) plotted as a function of mol% SP B 59 80 in 4:1 DPPC:POPG and 4:1 POPC:POPG liposomes, which are separated according ................................ ................................ .................. 130 6 8. Depth parameter plotted as a function of mol% SP B 59 80 in 4:1 DPPC:POPG and 4:1 POPC:POPG liposomes, which are separated according to the ................................ ................................ ................................ 131

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16 LIST OF ABBREVIATIONS AAA Amino acid analysis ARDS Acute respiratory distress syndrome B 0 External magnetic field Bis Tris 1 3 Bis[tris(hydroxymethyl)methylamino]propane CCD Charge coupled device CD Circular d ichroism chol Cholesterol CLD Chronic lung disease CLSE Calf lung surfactant extract CRD Carbohydrate recognition domains CW Continuous wave D Diffusion coefficient DLS Dynamic light scattering DPH Diphenylhexatriene DPPC 1,2 Dipalmitoyl sn glycero 3 phos phocholine DSC Differential scanning calorimetry E Energy EDTA 2,2',2'',2''' (Ethane 1,2 diyldinitrilo)tetraacetic acid E L Left circularly polarized light EPR Electron paramagnetic resonance E R Right circularly polarized light ER Endoplasmic reticulum FDA Food and drug administration FRET Fluorescence resonance energy transfer

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17 FT IR Fourier transform infrared HEPES 2 [4 (2 hydroxyethyl)piperazin 1 yl]ethanesulfonic acid HPLC High performance liquid chromatography IR Infrared k KL 4 KLLLL KLLLLKLLLLKLLLLK L Liquid crystalline phospholipid phase L Gel phospholipid phase LB Lamellar body LBPA Lyso bis phosphatidic acid LED Light emitting diode LPC Lyso phosphatidyl choline LS Lung surfactant LUV Large unilamellar vesicle m Molar ellipticity MD Molecular dyn amics MLV Multilamellar vesicle MOS Metal oxide semiconductor NiAA Nickel (II) acetylacetonate hydrate NMR Nuclear magnetic resonance PA Palmitic acid PAH Polycyclic aromatic hydrocarbon PC Phosphatidylcholine PDB Protein data bank PE Phosphatidylethanolam ine

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18 PG Phosphatidylglycerol PI Phosphatidylinositol PL Phospholipid PMT Photomultiplier tube POPC 1 Palmitoyl 2 oleoyl sn glycero 3 phosphocholine POPE 1 Palmitoyl 2 oleoyl sn glycero 3 phosphoethanolamine POPG 1 Palmitoyl 2 oleoyl sn glycero 3 [phosphor r ac (1 glycerol)] PPDPC 1 Palmitoyl 2 (pyrene 1 yl)decanoyl sn glycero 3 phosphocholine PS Phosphatidylserine PSPC 1 Palmitoyl 2 stearoyl sn glycero 3 phosphocholine PTFE Polytetrafluoroethylene Q Quantum yield QTH Quartz tungsten halogen r Radius R Univers al gas constant RDS Respiratory distress syndrome rER Rough endoplasmic reticulum R f Retention factor SAP Surface associated phase SDSL Site directed spin labeling SFM Scanning force microscopy SIMS Secondary ion mass spectrometry SM Sphingomyelin SP A Su rfactant protein A SP B Surfactant protein B

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19 SP B 1 25 FPIPLPYCWLCRALIKRIQAMIPKG SP B 59 80 DTLLGRMLPQLVCRLVLRCSMD SP C Surfactant protein C SP D Surfactant protein D ssNMR Solid state nuclear magnetic resonance SPPS Solid phase peptide synthesis SUV Small u nilamellar vesicle T Absolute temperature Tm Phase transition temperature TM Tubular myelin TMA DPH Trime thylammonium diphenylhexatriene TOF Time of flight Tr Trace USA United States of America UV Ultraviolet Vis Visible WHO World Health Organization

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20 Abs tract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy MONITORING THE EFFECTS OF SYNTHETIC LUNG SURFACTANT PEPTIDES ON LIPID BIL AYER DYNAMICS AND FLUIDITY By Otonye Braide May 2014 Chair: Gail E. Fanucci Major: Chemistry Premature infants with underdev eloped lungs typically develop respiratory distress syndrome (RDS) because their lungs lack the surfactant lining crucial for ox ygen absorption or have genetically failed to produce critical components of lung surfactant (LS) needed for proper function. The LS is a complex mixture of lipids and proteins known to provide a protective barrier against inhaled pathogens, lower alveolar surface tens ion and promote oxygen exchange. T he functional significance of LS is clear; however, a detailed mechanistic understanding of how lipids are trafficked to and from the air fluid interface for oxygen absorption remains unknown. Though the bulk of LS is made up of lipids (~90%), it is non functional without the presence of surfactant proteins (SP A, B, C, and D), especially SP B which is known to reduce surface tension and closely associates with lipids in the bulk phase of liquid below alveoli surface film (hypophase) As SP B is highly hydrophobic and structurally complex challenges in synthesis and expression of a functionally active recombinant have led to increased efforts to use synthetic alternatives in developing novel therapeutics for RDS treatment, and to elucid ate the mechanism of function. Within the scope of this dissertation, we use alternatives such as KL 4 a mimetic of SP B, and SP

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21 B 59 80 ; which have been shown to retain the fundamental properties necessary for LS function when i n the presence of major phospholipid constituents as model systems to further our understanding of fundamental membrane protein interactions and of specific LS components function Fluorescence and electron paramagnetic resonance techniques were used t o investigate the effects of the synthetic peptides on lipid dynamics fluidity and to model peptide depth profiles in lipid bilayers. The results from these experiments suggested a deeper penetration of the peptide into lipid systems that are rich in t he m ain lipid found at the air fluid interface monolayer ; and also showed induction of curvature strains in the bilayer both of which correlate with the hypothesized mechanism of lipid flipping to the air fluid interface Circular dichroism was used to evalua te pH effects on peptide conformation which highlighted the importance of using biologically relevant conditions to accurately model the molecular biophysics of LS.

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22 CHAPTER 1 INTRODUCTION AND RESEARCH OVERVIEW In 2012 the World Health Organization (WH O) reported 15 million babies per year are born premature ly worldwide, of which over a million d ie due to complications 1 In h the United States of America (USA), where 1 in 8 are born premature (Figure 1 1) the annual prevention and care costs associated with these complications are estimated at $26.2 billion annually; and w ith preterm birth rates inc reasing, medical and educational efforts have shifted towards research and innovation in hopes to reduce the se expenses. A particular area of interest for these efforts has been in the treatment of respiratory distress syndrome (RDS), a leading cause of mo rbidity and mortality in preterm infants 2 RDS is a breathing disorder that is attributed to a deficiency in the production of lung surfactant (LS), or its critical components which are needed for proper function. Currently, the most wi dely u sed method of treating RDS involves the administration of mammalian based LS extracts, which in creases the risk of immunogenic and infectious complications For this reason, and to reduce long term costs, novel therapeutic agents are needed and the m echanism of LS function is not known. In this dissertation, various spectroscopic techniques will be utilized to elucidate the mechanism of LS function. Lung Surfactant To understand the importance of lung surfactant, it is crucial to understand how the r espiratory system works. The pulmonary system (or respiratory system) is a network of various organs and structures enabling efficient and rapid gas exchange between an organism and its environment Particularly in mammals, oxygen diffuses into blo od vesse ls and the heart pumps the oxygenated blood to the rest of the body for

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23 distribution into cells. This process begins in the lungs, specifically in the alveoli, which are surrounded by capillaries for ease of gas diffusion into and out of the blood stream 3 The small sacs of alveoli are highly curved increasing the surface area by approximately 300 cm 2 per 1 cm 3 of lung tissue which inherently maximizes the capacity for oxygen intake and the consequent expulsion o produ ct. However the highly curved feature of alveoli results in significant pressure differences within the lungs that must be alleviated to prevent collapse. In 1929 von Neergaard published a paper where he noted the pressure required to fill lungs with air varied depending on the presence of liquid 3 He also stated that the liquid layer, which lined the alveoli, helped stabilize them by lowering the naturally high surface tension of the air/water interface 3, 4 By the 1940s, a group of scientists reported finding a high content of the fully saturated, 1,2 d ipalmitoyl sn glycero 3 phosphocholine (DPPC), in lung tiss structures (alveoli) contained a fluid, surface active material, also known as surfactant, whose quantity and quality contributed to their stability 5 In 1957, Clements did extensive surface tensions measurements on films extracted from lungs, showing the ability of the surfactant to modify the elasticity and having a clear effect on the lung mechanics, however, he was unable to d etermine its effect on the diffusion across the alveolar barrier 6 Both Pattle and Clements noted the surfactant adsorb s and quickly spread s forming an insoluble film 7 During this period, it b ecame clear that the lipid content of this fluid material was playing a crucial role in the spreading, and overall effects measured in terms of lowered surface tension 3, 8 In 1959, A very and Mead drew a direct correlation between surfactant deficiency and the increased surface tension

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24 measured in lung extracts from underdeveloped infants and those dying with hyaline membrane disease. This stimulated an interest to further i nvestigate the lung surfactant (LS) and functional role of its components 9 Life Cycle and Function The LS is an extracellular material whose components are first synthesized intracellular ly in type II pneumocytes (Figure 1 2 ) 10 which are cells found in the alveoli fluid layer. The process begins in the rough endoplasmic reticulum (rER) and Golgi apparatus from which a ll the essential lipid and protein components are transported and then densely packed into bilayers within storage granules called lamellar bodies (LB) ; 11 t he co mposition present in LBs have been shown to be near identical to surfactant obtained from bronchoalveolar lavage 12 13 Further studies us ing electron microscopy have also shown LB secretory function as it is released into the alveolar subphase (hypophase region). The LBs expand and transform into a complex lattice like structure; an ordered ne twork called tubular myelin (TM) from which ra p id adsorption of material to form an interfacial lipid film 14 at the air water interface occurs during inspiration. The components of this precursor material are trafficked to form a surface asso ciated phase (SAP) which consists of vesicular structures linked to a monolayer of lipids at the air water interface. The direct mechanism by which this monolayer is generated is not known nor are the specific details of how lipid components are moved to various phases to form pertinent structures hence the motivation of research efforts During breathing cycles, specifically with expiration, compression of the interfacial film occurs, which simultaneously lowers the surface tensio n at the interface and promotes alveolar stability. Though there are two varying theories on the specific details of the life cycle within the subphase, it is known that DPPC rich and poor

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25 particles from the surface associated phase (SAP) and monolayer film at the interface, ar e retrieved by the type II pneumocytes for recycling into new LBs. Alveolar macrophages enable the degradation of surfactant components, though the pathway differs for phospholipids and proteins due to selective recognition based on structure 15 3 16 As suggested by CC Macklin in 1954, type II pneumocytes play a significant role in secretion of the surfactant for various functions such as lower ing surface tension in the alveoli to prevent collapse of the lungs and e nable expansion, aids in the removal of inhaled particles, acts as an agent to prevent the reproduction of bacteria, as well as helps prevent extracellular fluid from entrance into the alveolus 4 Specifically the hydrophilic proteins SP A and SP D have been linked to host defense in the respirator y tract, while SP B and SP C are known to aid in the surface tension regulation and lipid trafficking. The phospholipids are necessary for forming the surface active film at the air water interface but they also function as the platform for assembly of sur factant structures 5 Composition LS is a complex mixture of approximately 90% lipids and 10% proteins by weight 17 18 15 which is produced by type II pneumocytes (alveolar type II cells) 19 20 Approximately 80% of the lipids are phospholipids, of which ~50 to 70% are phosphatidylcholines, the bul k being the fully saturated DPPC, and the anionic POPG makes up ~10% 15 (Figure 1 3) T ypically, such high levels of DPPC are considered exclusive to LS, however brain myelin and erythrocyte membranes contain relatively high levels with DPPC accounting for 10 20% of the pho sphatidylcholine (PC) present 7, 21 Some of the minor phospholipids include monounsaturated PC,

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26 phosphatidyl ethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), lyso phosphatid ylcholine (LPS) and sphingomyelin (SM) Other lipid components include tryacylglycerols, cholesterol, and free fatty acids. From a number of experiments on surfactant extracted from different mammalian species (mice, rabbits, sheep, etc. ), t he ratio of the se lipids (Table 1 1) and their general localization within structures tend to vary A pproximately 8 10% are the surfactant associated proteins: surfactant protein A (SP A), B (SP B), C (SP C) and D (SP D). These proteins are typically categorized by their polarity and molecular weight: SP A and SP D being hydrophilic and large molecular weight SP B and SP C being hydrophobic and low molecular weight Lipid Structures and Function s Lipids are organic molecules that are generally hydrophobic or amphiphilic in nature. It encompasses a number of compound groups such as fats, waxes, vitamins, glycerides, terpenes, steroids, etc. In eukaryotes, lipids are classified according to their specific biological function such as energy storage, signaling, and being com ponents of cell membrane structures (F igure 1 4 ) Though all types of lipid classes are present in LS the majority are phospholipids, which form bilayer structures that are crucial in organizing 22 intracellular organelles, and in the movement of components betwee n intracellular and extracellular compartments. Phospholipids are amphipathic with two fatty acid chains and a phosphate esterified to a glycerol backbone (Figure 1 5 ) The fatty acid chains (acyl chains) are located at the sn 1 and sn 2 positions of th e backbone and are referred to as hydrophobic tails, while varying polar headgroup s are attached to the phosphate located on the third carbon of the backbone. The standard nomenclature for phospholipids is dependent on the general characteristics it posses ses. For example 1 palmitoyl 2

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27 oleoyl sn glycero 3 phosphocholine or POPC, the first two letters indicate the fatty acid chain lengths and degree of saturation, palmitoyl being 16 carbon length chain, oleoyl being 18 carbon chain length and 1 site of unsat uration. The last two letters indic ate the phosphate and choline functional groups ( headgroup ) making this particular lipid a zwitterionic or neutral lipid due to its net charge being zero ( Figure 1 3 ). The chain length, degree of unsaturation and headg roup affect the ph ysical properties of each lipid; the collective effect on these properties also depends on the interactions between lipid mixtures Hydrogen bonding between headgroup s and van der waals forces between the acyl chains contribute to the mel ting temperature; temperature at which the phospholipids go from gel (L ) to fluid (L ) state. As the chain length increases the melting temperature also increases. The presence of double bonds (degree of unsaturation) in the acyl c hains cause disruptions in the forces between hydrophobic chains causing a decrease in the melting temperature, hence the large difference between DPPC and POPC with T m of 41 C and 2 C respectively. Considering the surfactant is a mixture of ~40% DPPC, ~30% POPC, ~10% POPG, o ther lipids and proteins, the T m has been shown to fall between 34 and 36 C, below physiological temperatures. T he presence of multiple monounsaturated modifies the physical properties of LS particular in increasing fluidity, which helps in the spreading dynamics of the surfactant film across the alve olar surface. As mentioned previously, t he amphipathic nature of surfactant lipids is crucial to their role in LS function As the headgroup s are hydrophilic and the tails are hydrophobic, the environment t o which they are exposed causes them to self assemble into various structures, otherwise referred to as polymorphism. Specifically, when

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28 phospholipids are immersed in polar environments, their arrangement is driven by the concentration and pacifying the en tropy experienced described by the hydrophobic effect. At low concentrations, the lipids exist in their monomeric form, meaning each molecule is fully solvated by water molecules. However, as the concentration approaches it critical micelle concentration ( CMC), maintaining the monomeric form becomes less energetically favorable as water must be more ordered, lowering the entropy, hence self asso ciation becomes more prevalent. At this point, the steric and ionic repulsive forces of the polar region is lower than the attractive forces of the non polar region leading to organization into structures such as micelles, hexagonal phase normal, bicelles, lamellar phases, etc. (Figure 1 6 ) In particular, when surfactant material forms the surface associated phase, i t is known to exist as lipid vesicles (or liposomes) (Figure 1 7 ) This arrangement establishes a hydrophobic barrier that is not easily penetrated by molecules of dissimilar polarity. The dynamic forces and biophysical properties of phospholipids contribu te to their functional behavior at the surface. Initial studies to understand the roles of specific lipids in surfactant function utilized Langmuir Wilhelmy balance, pulsating bubble surfactometer, and captive bubble tensiometer to investigate the effec ts of individual components on surface tension reduction a necessity to prevent lung collapse. Clements 6 theoretical calculations suggested low surface tensions approaching 0 mN/m were necessary to maintain lung stability during expiration, hence la ter experiments w here DPPC films lowered equilibrium surface tension from ~25 mN/m to less than 2 mN/m with area reductions of 10 12% supported his predictions 7 In comparison, unsaturated PC films we re only able

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29 to lower surface tension to 15 20 mN/m with dynamic compression which can be attributed to their inability to form well ordered (or tightly packed) layers due to the double bond present in the acyl chain, and the significant difference in tra nsition temperature. The experiments also showed a r apid return to equilibrium values once compression ceased, unlike DPPC films that took hours to return to equilibrium. This data supports the need for a monolayer rich with DPPC at the air water interface However, DPPC pure films which are known to have a T m of 41 C, would be extremely slow to adsorb to form the surface active monolayer. Hence other studies have shown that the presence of unsaturated or neutral lipid components is crucial for enhancing t he adorption 7, 23 Acidic phospholipids such as PG and PI typically make up 8 15% of the surfactant composition. Interestingly, the ratio of PG and PI vary significantly when comparing adult surfactants to fetal or neonatal surfactants; PG is lower while PI is elevated in the fetal and neonatal surfactants. Mixing these fluid acidic lipids with DPPC has been shown to enhance adsorption, in particular 1 Palmitoyl 2 oleoyl sn glycero 3 [phosphor rac (1 glycerol)] P OPG, contribute s to the spreading ca pabilities of the surface film, while o ther minor phospholipid components are predicted to be involved in signaling events associated with surfactant metabolism. The addition of neutral lipids such as free palmitic aci d (PA), were shown to enhance the rate of adsorption in both DPPC and DPPC PG layers presumably by introducing minor packing defects in the membrane structure. Cholesterol also being a constituent that enhance s ad s orption of DPPC rich vesicles, has been sh own to decrease packing 23 increase fluidity and film respreading 24 2 5 As mentioned previously,

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30 increased fluidity during compression is not favorable in lowering surface tension 26 27 therefore its presence at the interfacial monolayer film would have to be minimized. Various reconstitution studies 28 where they studied the surface activity of in vitro mixed lipid films, have shown that the most functionally effectiv e preparation included DPPC, PG PA, and hydrophobic proteins; these mixtures promoted lung expansion in prematurely delivered rabbit pups of 27 days gest ation. Protein Structures and Functions As mentioned previously, there are four surfactant proteins: SP A, SP B, SP C and SP D (Figure 1 8 ) SP A, SP B, and SP C are typically obtained in bronchoal veolar lavage and shown to be closely associated with LS membranes. SP A and SP D are hydrophilic and consist of large macro molecular assemblies, for example the quaternary structure of SP A consists of a hexamer of trimers, which contain carbohydrate recognition domains (CRD). 23 SP A is able to bind a multitude of ligands such as sugars, Ca 2+ and phospholipids; it is also essential for the formation of TM after secretion of LBs into the hypophase. Both SP A and SP D are known to specifically bind to bacteria, fungi and viruses, enabling reco gnition and removal. This allows LS to function as a protective barrier against inhaled substances ( host defense ) SP B and SP C are hydrophobic proteins which are closely associated with the surfactant lipids, are critical for lipid organization and surf actant durability 29 directly impacting surface tension reduction. 30 Both relatively small proteins (low molecular weight) and present in low concentrations, they are necessary for proper film formation. Gene knock out studies characterized SP B as an respiratory failure occurs in its absence 31 32 Particularly SP B is shown to be necessary for the production of surfactant and its packing into LBs. In vitro studies have shown it to

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31 be ef ficient in transferring surface active phospholipids 23 from mem branes to the interfacial film and that it remains permanently associated with lipid membranes be ing distributed in regions of disorder. It is also shown to perturb packing in membranes and films, which can lead to leakage of vesicle contents, phospholipid exchange between membranes and membrane fusion. These perturbations suggest its role in the mech anism of lipid trafficking. Though SP C, the smallest of the surfactant proteins, has not been categorized as crucial for respiratory function, deficiencies in expression can lead to other severe respiratory diseases. Unlike the planar configuration of S P B in membranes, SP C has been determined to ado pt a transmembrane orientation, however it also partitions into regions of disorder. It is able to aid in mechanistic transfer of phospholipids between membranes but is mainly know for promotion of interfaci al lipid film formation. Respiratory Conditions and Treatments A s mentioned previously, 15 million infants are born premature worldwide annually. In the USA, one in eight babies are born premature, of which 10% will develop respiratory distress syndrome (RDS) due to structural immaturity of the lungs or genetic failure to produce the essential SP B. Due to the nature of this disease, many research efforts have been focused on the development of surfactant replacement therapies. In the 1960s, initial expl orative treatments employed aerosol administration of DPPC shortly after the phospholipid was discovered to be a major component 8 However, the negative effects observed during the studies led to the conclusion that oth er components were crucial for L S functionality. Following these trials, researchers began to turn to mammalian sources. When noticeabl e improvements in pulmonary mechanics were observed in rabbit pups treated

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32 with extracted adult rabbit lung surfactant, methods of extracting a wide range of animal based surfactant were optimized and the significance of the protein components were tested. The use of exogenous natural surfactant from bovine lungs enriched with synthetic lipids, along with ventilator support, was the first reported treatment of newborn infants with RDS proven to be successful 8, 33 To date, bovine derived surfactants are the most widely used form of treatment for various chronic lung diseases (CLDs) 8 33 However, growing concerns about the potential risk of immunogenic and infectious complications 34 expense, supply limitations, and inconsistencies in surfactant composition due to extraction methods, have inspired research efforts towards the development of purely synthetic options 35 36 37 38 39 40 P revious studies determined that the major phospholipid component (DPPC) 8 can not maintain L S functionality, demonstrating the necessity of lung surfactant proteins (SP A, SP B, SP C and SP D) 41 42, 43 However, e xpression of the essential lipid membrane associated proteins, SP B and SP C, in their native form poses a challenge due to their hydrophobicity and lack of stability, particularly with SP C 44 This has led to the construction of various synthetic analogs : mini B, SP B 1 25 SP B 59 80 KL 4 SP C33, etc 43 45 KL 4 43 46 47 a major component of Surfaxin 48 49 50 51 52 53 is the first synthetic peptide containing surfactant 54 to be approved for use in neonatal medicine by the Food and Drug Administration (FDA) in 2012 It was designed to mimic the C t erminal of SP B. Though its amino acid sequence is significantly different from the protein (Figure 1 9 ) similarity in its charge density and hydrophilic/hydrophobic ratio 55 enables retention of functionality

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33 Research Overview The structural con servation of SP B C terminus by KL 4 and its ability to maintain surfactant functionality with limited amino acid overlap, make it of particular interest to further our understanding o f membrane protein interactions within biological systems Despite the a dvances in elucidating structural properties of the synthetic surfactant peptides, molecular level information is still pertinent to understanding how it moderates surface tension reduction and interfacial film fluidity in the a l veoli and to elucidate the mechanism by which lipid trafficking occurs In the scope of this dissertation, we aim to further elucidate the mechanism of LS function by applying various spectroscopic techniques: CD, fluorescence 56 57 58 59 and EPR, to study localized environmental changes and lipid dynamics Our goal is t o investigate the effects of KL 4 and peptides of SP B, structure and orientation on phospholipid b ilayer dynamics and fluidit y. We have inc orporated a pyrene labeled phospholipid analog (PPDPC) in our chosen lipid systems, 4:1 POPC:POPG and 4:1 DPPC:POPG, and expect changes in the membrane free volume with peptide integration to modify the pyrene excimer to monomer ( I e /I m ) ratios as a direct correlation of effects on lateral mobility and changes in the local concentration of the fluorophore within the bilayers 60 The fluorescence anisotropy of fluorphores (DPH, TMA DPH, a nd r hodamine PE), were used to monitor changes in fluidity properties within lipid phases. The rhodamine PE probe was used to monitor surface activity upon peptide binding to possibly track phase separation within bilayer systems. These techniques present advantages over other high resolution techniques considering the experimental time scale and low sample consumption, making it a cost effective option

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34 Figure 1 1 Estimated rates of preterm births in 2010 (adopted from Wo rld Health Organization Born Too Soon: The Global Action Report on Preterm Birth ). 124

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35 Figure 1 2 Lung surfactant life cycle ( http://www.jped.co m.br/conteudo/01 77 S3/ing_print.htm#1 )

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36 Figure 1 3 Chemical structures of main glycerophospholipids found in surfactant: A) DPPC B) POPC and C) POPG. Table 1 1. Lipid composition of extracellular surfactant in selected ma mmalian species by weight. (Adapted from The role of lipids in lung surfactant by Ruud Veldhuizen et al 7 ) Phospholipid composition (%total) Cholesterol [%disatu rated] (%chol/PL) PC LPC SM PG PI PS PE LBPA Mouse 72.3 0 3.3 18.1 1.9 9.7 Rat 82.3 [49.3] 0.3 0.8 7.5 [32.3] 1.8 [2.2] 0.1 5.1 Tr 7.1 Rabbit 80.6 [52.6] Tr 1.5 7.2 [38.7] 4 [2.5] 1.9 4.4 Tr Ovine 81 Tr 1.7 7.9 2.6 Tr 4.8 2 Bovi ne 79.2 [49.9] Tr Tr 11.3 [33.3] 1.8 Tr 3.5 2.6 3 Human 80.5 [47.7] Tr 2.7 9.1 2.6 0.9 12.3 7.3 Tr = Trace amounts

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37 Figure 1 4 General classification of eukaryotic lipids (modified from Nelson and Cox) 61

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38 F igure 1 5 Chemical structures of A) glycerol B ) fatty acid C) an example of an acyl chain (blue) and D) glycerophospholipid where R represent s an acyl chain of 3 or more carbons and X is a headgroup (choline, glycerol, etc. ) Figure 1 6 Lipid polym orphisms : fluid (L ), gel ( L ), hexagonal ( H I ), inverted hexagonal (H II ) and various isotropic phases. 62

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39 Figure 1 7 Morphology of different sizes/types of liposomes in 2 dimensions

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40 Figure 1 8 Surfactant proteins A C and D models were generated in MacPymo l ( Schrdinger, LLC) ; SP B was adopted from Olmeda et al 63

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41 Figure 1 9 Sequence alignments for SP B, SP B 1 25 SP B 59 80 and synthetic peptide mimic KL 4 showing charged residues highlighted; basic in blue and acidic in red

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42 CHAPTER 2 INSTRUMENTATION THEORY AND METHODS USED Introduction Both multilamellar vesicles (MLVs) and large unilamellar vesicles (LUVs) composed of surfact ant lipids and protein s were utilized throughout as membrane mimetic systems thought to best replicate the subphase extracellular environment of the alveoli. S tandard protocols were utilized for all liposome preparations. Prior to preparing liposomes, the structural integrity and concentrations of all purchased lipid samples were determined by thin layer chromatography and malachite green phosphate assays which is a color i metric assay Differential scanning calorimetry (DSC), a thermoanalytical technique, was used to monitor any possible pe rturbations in the thermophysical properties of the liposomes that may have been caused by incorporation of the fluorescence probes. Circular dichroism (CD) was used to monitor the secondary structure conformation of pept ides with varying pH. F luorescence spectroscopy was employed to characterize changes in bilayer dynamics and fluidity before and after surfactant peptide assimilation. Power saturation electron paramagnetic resonance (EPR) was used to investigate the surfa ctant peptide penetration depth and effects on lipids at different positions along the bilayer such as the headgroup, plateau and hydrophobic core region. The timescale of lipid motions and the type of molecular interactions being evaluated determined the selection of fluorescence and EPR spectroscopy techniques. Thin Layer Chromatography Chromatography is a method used to separate mixtures based on molecular properties such as size, polarity and charge ; it is also used for the purification of

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43 compounds T hin layer chromatography (TLC) separate s compounds that partition differently between a stationary and mobile phase based on polarity T he stationary phase is a plate or sheet of glass, plastic or aluminum which is coated with an adsorbent layer made up o f silica gel, aluminum oxide, or cellulose ; t he mobile phase is a solvent phase that migrates up the TLC plate by capillary action T he mixture is spotted onto the TLC plate and as the mobile phase begins migrating upward, it passes thr ough the stationary phase and solubilizes the mixture, causing the solutes to also migrate. The rate of migration for each solute reflects the preferential interaction with either phase because there is a dynamic exchange between adsorption to the stationary phase and dissolu tion in to the mobile phase Each solute within the mixture will have a partition coefficient unique to its chemical structure leading to differences in re tention and inevitable separation on the s tationary phase. The plate is removed from the solvent phas e, air dried and prepped for visualization by charring, iodine vapor or fluorescence ; the spots become visible and the final migration distance of the mobile phase and of each solute is measure d to compute the retention factor ( R f ). (2 1) For our purposes, thin layer chromatography (TLC) is used to assess the structural integrity of purchased phospholip ids prior t o experimental us e In Figure 2 1, the chromatogram shows the separation of mixed phospholipids systems, DPPC:P OPG and POPC:POPG, relative to lyso phosphati dylcholine (LPC ); the negative control The LPC represents the degraded form of a phospholipid with only one acyl chain and the zwitterionic choline heagroup. 64 65

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44 Differential Scanning Calorimetry Differential scanning calorimetry (DSC) is a thermoanalytical technique used to measure phase behavior of stab le macromolecula r interactions. As mentioned in Chapter 1, lipids can self assemble into various polymorphic forms depending on their structure, the degree of hydration, temperature, pressure, ionic strength and pH; and each lipid phase ca n transition into another phase. 66 To in terrogate the effects of extrin sic fluorescent pro bes on the physical characteris tics of liposomes comp osed of the major surfactant lipids DSC was utilized for a comparative analysis of the lipid fluorescent probe sample to a reference sample. Over time, the temperature of both the reference and sample cell, was increased or decreased at a constant rate ( dT/dt ) and the differential rate of heat flow into the sample relative to the reference is measured The difference in t he energy required to maintain the two cells at the same temperature provides unique information when a point of heat release (exothermic) or heat absorption (endothermic) occurs. In the case of liposome analysis, the phase transition represents the melti ng event which occurs as the sample goes from gel to fluid phase ; the specific phase transition temperature ( T m ) is the point of 50% conversion This change directly correlates to the destabilization of molecular interactions as heat is absorbed, specific ally with liposomes, weak van der Waals forces found in the acyl chain regions of bilayers (hydrophobic core) begin to dissociate. However, the strength of electrostatic interactions and hydrogen bonding also begins to dissipate. DSC gives the properties o f enthalpy and heat capacity for given states of change. By integrating the thermogram peak, followed by baseline correction, the calorimetric enthalpy can be extracted and directly shows the energy uptake by the sample. By measuring the transition enthalp y

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45 as a function of melting temperature the v an Hoff enthalpy ( H vH ) parameter can be obtained which allows determination of the nature of the transition as shown by the following equations: (2 2) (2 3) (2 4) w here S is change in entropy in cal/K 1 mol 1 H cal is the calorimetrically determined enthalpy in kcal/mol, 1/2 is the temperature width at half height, H vH is used to calculate the cooperative unit size ( CUS ); the intermolecular cooperation between p hospholipids. Spectrosco py Spectroscopy is the study of the interaction of electromagnetic radiation with matter. The spectroscopic method used to produce, measure and interpret spectra generated from these interactions, depends on the analyte species, th e interaction type (ex. absorption, emission, diffraction), and the region of the electromagnetic spectrum used in the analysis (Figure 2 2) Whether quantitative or qualitative, each method provides information about distinct molecular or atomic transitio ns, which occur as a result of exposure to radiative energy. The basis of these transitions requires a detailed understanding of the dual nature of light: particulate and wavelike (Penner et al. Food Analysis book) Light Light is described as particles o f energy that move through space with wavelike properties. The energy distributed is dependent on the propagation of associated

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46 electric and magnetic fields leading to phenomena such as interference, diffraction, and refraction. It is also known that the e nergy is compiled in discrete packets along the perpendicular components (electric and magnetic fields), rather than continuous through space. Though similar to particles of matter (electrons, neutrons, protons) which also display wavelike properties, ligh ts interaction with matter is best characterized by its particulate nature in further understanding absorption and emission processes. E lectromagnetic radiation can be described in terms of wavelength, frequency and amplitude with respect to the plane of polarization and direction of propagation. As shown in Figure 2 3 t crest) of a given wave and the units depend on where the method falls on the electromagnetic spectrum. Some methods report t hese values in terms of wavenumbers, which is the inverse of the wavelength in centimeters. The frequency is the number of oscillation cycles the wave will make within a given period; units are per second. The amplitude is essentially the height (or magnit ude) of the wave at the maxima. The velocity of propagation is the distance per second a wave travels within a given medium. Contrary to the frequency, which depends on radiation source and stays constant in different media, the velocity of propagation var ies, impacting the wavelength of radiation. The power and intensity of radiation are proportional to the square of the amplitude of the associated electromagnetic waves which is made of oscillating electric and magnetic fields that are in phase and perpen dicular to each other. Obtaining a purely electric or magnetic component for spectroscopic analysis is impossible; instruments are designed to select for one component while significantly minimizing the effects of the other.

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47 As light is a moving (or tran sverse) wave that consists of oscillations occurring perpendicular to the direction of energy transfer, photons are used to describe and quantify the particulate nature of that energy. More specifically, as shown in the E quation 2 5 the energy of a photon is directly proportional to t he frequency of associated wave; where E is the radiative energy, h is the (2 5 ) frequency, c is the speed of light in vacuum (approximately 3 10 8 m/s), an d is the wavelength. Given that the frequency of a wave is constant, the energy of the photons making up monochromatic light also stays constant. Matter Most atoms and molecules exist at the lowest energy state referred to as the ground state. However, irradiation from various sources (ex. electromagnetic, acoustic, pressure, etc. shift to higher energy states. These energy states are in discrete steps, not continuous, due to the quantum nature of energy levels that become signature to distinct species being analyzed. These energy levels are categorized into the electronic, vibrational and rotational levels. The potential energy for ele ctrons in different orbitals yields to differences seen in electronic states during absorption and emission processes as shown in the molecular energy level diagram (Figure 2 4) The vibrational levels are representative of mol ecules being in constant moti on, though s ome vibrational relaxation effects may be a result of the external environment (ex. s olvent effects). The rotational levels capture the rotation of a

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48 molecule about its center of gravity. The energy of the photons associated with these various levels requires different spectroscopic method to capture these transitions. UV Vis Spectroscopy When evaluating molecules such as transition metal ions, highly conjugated organic compounds, a nd biological macromolecules, UV Vis spectroscopy is employed to record absorption events that yield electronic transitions from the ground states to the excited states (Figure 2 4) The energy gained must be sufficient to excite molecular electrons from the highest occupied molecular orbital (HOMO) to the lowest uno ccupied molecular orbital (LUMO). For example hydrogen s orbitals are known to have spherical symmetry around a single nucleus, and when two nuclei come within equilibrium bond length, an overlap in their wavefunctions can lead to the formation of the diat omic hydrogen (H 2 ). This sigma bond formation is a product of constructive interference; having an additive effect on the overall electron density shared between the nuclei. Based on the molecular orbital theory, the 1s orbital (or bonding orbital), is depicted as having a lower energy than two separate hydrogen atoms (Figure 2 5) This energy state typically contains two electrons of opposite spin yielding a covalent bond. The antibonding orbital is essential a destr uctive interference where the wavefunctions cancel each other out upon overlap. As other atomic spe cies have more electrons available for bonding, the complexity and variety of possible t ransitions increases (Figure 2 6 ). In all cases, an external source o f energy is required to pacify the energy gap between transitions. When a sample is irradiated and encounters light with energy that matches electron promotion to a higher energy orbital, the spectrometer records the wavelengths at which absorption occu rs and the level of intensity. The intensity of the absorbance

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49 will directly correlate to the number of molecules present hence a corrected absorption value, molar absorptivity, is used in comparing different compounds. Molar absorptivity is governed by th e Beer Lambert law (Equation 2 6 ), where A is (2 6 ) absorbance, C is the sample concentration in moles per liter, l is the path length in cm. Molecules that absorb light in the 200 to 800 nm are known to be chromophores containing pi electron systems or hetero atoms with non bonding valence electron shells ( Table 2 1 ) 67 Spectrophotometer For a standard spectro photo meter the instrumentation components necessary for measurements, can be compartmentalized into the light source, wavelength isolator sample holder, and a detector. The ideal set up would consist of a light source with constant emission intensities at all wavelengths, a monochromator that can filter for specific wavelengths without bias, and a detector that is equally sensitive at all ranges. In a dual beam spectrometer (Figure 2 7 ) the deuterium lam p is continuous in the ultraviolet range, while the tungsten filament is useful in the visible range. Other types of light sour ces include: xenon arc lamps, light emitting diodes (LEDs) etc The monochromator (or prism) is made up of diffraction gratings with etched grooves, which enable separation of the incident light into various wavelengths, which is selectively applied to sample to scan for the absorption process. Ideally, if samples could be placed within path of light, with air as a medium and zero pa rticulates to obstruct the path; there would be minimal interference and error with detected signal. H owever, each sample is typically solubilized and placed into a holding cell known as a cuvette. The solvent in which the sample is dissolved can also ab sorb causing deviations from the ideal and

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50 though cuvettes are generally transparent, or at least the region that comes in contact with light is; e ach type of cuvette has its own set of optical properties, therefore will absorb within specific wavelength r egions (Figure 2 8 ). Cuvettes are typically made of plastic, glass or quartz. Therefore choo sing a cuvette is highly depende nt on the sample range of absorption. P hotomultiplier tubes (PMTs) are commonly used for detection and are composed of a photocatho de, dynodes and an anode. When photon s emitted from the sample hit the photocathode, which is an electrode coated with a photosensitive compound, the absorbed energy induces the ejection of electrons from the m etal film ; this process is known as the photoe lectric effect. The electrons are focused and then multiplied by secondary emission events as they are passed down a series of electrodes, the dynodes, by incremental potential differences. Arrival of the electrons at the anode results in a detectable curr ent pulse. Photodiodes and charge couple devices (CCDs) are alternative form s of detection both involving the formatio n of an electron hole pair upon photon impact. Photodiodes depend on the wavelength of light to come in contact with the diode array from the monochromator ; this cause s the migration of the electrons and holes producing a photocurrent, this is the inner photoelectric effect. The CCD array is referred to as a metal oxide semiconductor (MOS) capacitor, in that it is able to conduct the direct ion of electron hole movement by an electric field bias being applied, but is also able to store charges. Signal Output The light source with equal intensity the absorbance is measured at varying wavelengths of the molecules being analyzed, the light inte nsity passing through the sample is compared to light intensity passing through a reference media. This reference

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51 media is se lected to be transparent (near zero absorption) therefore the light collected at detector is assumed to be approximately 100% of th e incident beam, also referred to as the initial intensity ( I 0 ). This value is compared to light intensity collected by the detector after passing through the sample ( I ); the ratio of these intensities is called transmittance ( I/I 0 ), and is expressed as a percentage ( %T ). The overall absorption is expressing in equation : (2 7 ) The samples are typically placed in cuvettes with an internal width of 1cm as suggested by the pathlength in Beer Lambert law equation. For optimal measurements fused silica or quartz glass cuvettes are used to eliminate absorp tion at the ultraviolet, visible and near infrared (IR) regions; though plastic cuvettes are used in some cases. This technique was used for malachite green phosphate assays in more accurate determination of lipid concentrations. Circular Dichroism (CD) Sp ectroscopy Similar to UV Vis spectroscopy, circular dichroism (CD) is a technique based on light absorption of samples as a function of scanned wavelength range. Specifically, the differences in absorption as measured from circularly polarized light inter acting with sample in opposing directions, left (rotating counter clockwise) and right (rotating clockwise) handed, are used to determine structural information about optically active molecules such as biological molecules. These molecules generally have c hiral centers which cause a change in the orientation of the polarized light by exerting torsional force on it the direction of rotation being influenced by the handedness of the molecule. The interaction of the left and right handed circular l y polarized light with chiral centers will yield difference in the magnitude of the electric field vectors, E L and E R respectively.

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52 Therefore, after passage through a sample, the resultant light becomes a combination of the unequal absorption of the circular polarize d lights yielding an elliptical polarized light (Figure 2 9 ) Polarized Light Incident light is composed of waves oscillating on multiple planes perpendicular to the direction of travel There in spectroscopic techniques, polarized refers to light waves oscillating in a single plane. In order to generate light in a single plane, various classical methods are used: reflection off a transparent media at a specific angle, reflection of glass mirrors, and passage through a prism. Assuming the waves are trave lling in direction z linearly polarized light would be the result of the horizontal ( x ) and vertical ( y ) wave oscillations being in the same phase. To generate circularly polarized light, the x and y waves, of equal amplitude are set a quarter wave ( ) out of phase with each other (Figure 2 10 ) Application CD is commonly used technique for qualitative determination of secondary structure elements in proteins (Figure 2 12 ) This is monitored in the far UV regions of approximately 180 260 nm wher e peptide bonds, amino acids with aromatic side chain s, disulfide bonds and prosthetic groups strongly absorb The structure of the environment in which the peptide bonds (chiral centers) is located will enable detection of absorption events of distinct tr ansitions: the n 200 nm (Figure 2 11 ) For a standard alpha helical protein characteristic negative bands are displayed at 222 nm and 208 nm with a positive band at 190 nm. Anti parallel beta sheets yield a negative band at 218 nm and posit ive band at 196 nm, while random coil structures, disordered proteins display a negative band at 195 nm and a small (low

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53 elli pticity) positive band above 212 nm. Raw data collected is typically in units of millidegrees and is converted to molar ellipticity ( ) as shown in equation below (2 8 ) This method was applied in understanding the effect of pH on peptide structure being analyzed. Fluorescence Spectroscopy As the molecular energy level diagram (Figure 2 4 ) suggests, absorption phenomena can be followed by emi ssion of heat and light from substances (Figure 2 13) which is known as luminescence. Depending on the nature of the excited state, it can be categor ized as fluorescence or phosphorescence phenomenon. In fluorescence, the electron in the excited state is paired to an electron at ground state by opposite spin, the singlet state, hence relaxation to the ground state is an allowable transition that occurs rapidly. While in phosphorescence, involves the pairing of electrons of the same spin in the excited and ground state, a forbidd en transition, therefore having a slower emission rate. The rate of emission, the average time it takes between point of excita tion and the return to ground state, is called the lifetime ( ). In fluorescence, t he rate of relaxation to the ground state via emission is typically on the order of 10 8 per second ; the average fluorescence lifetime being approximately 10 ns. Considering light travels 30 cm in a nanosecond, each fluorophore will di splay subna nosecond lifetimes, as a result, the instrumentation needed to capture these events require highly precise optics and electronics.

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54 Fluorometer The basic components of a spectrofluorometer are the light source, excitation and emission monochrom ators, sample c ell, and a detector (Figure 2 14 ). Similar to UV Vis absorption, there are a set of ideals at which each component is expected to perform: (1) the intensity of light emitted should be constant at all wavelength (2) the passage of light throu gh the monochromators must be equally efficient across all wavelengths (3) this efficiency must be independent of the direction of polarization and (4) detection of the photons must also be equally efficient at all wavelengths 68 However, despite careful machining of ea ch component, distortion s are introduced into spectra as a result of wavelength dependent efficiency of monochromators and the detection system. For a detailed description of how each component functions, the Jobin Yvon Horiba (Edison, NJ) FluoromMax 3 flu orometer will be used as a standard (Figure 2 15 ). In this specific case, the light source is a 150W Xenon arc lamp is its continuous light source. This light is collected by a diamond turned elliptical mirror and focused toward the entrance slit of the excitation monochromator. The reflection grating of the monochromator disperses the incident light using vertical grooves (1200 grooves/mm). Each groove is etched at a specific angle to optimize the gratings reflectivity. Etching was done at 330 nm (excita tion) for UV and Vis region, and at 500 nm (emission) for the high UV and near IR region. These gratings are coated with magnesium fluoride to prevent oxidation. All spectra are obtained by recording the intensity values at each wavelength, with the abilit y to scan up to 200 nm/s with an accuracy of > 0.5 nm and repeatability of 0.3 nm. Slit adjustments directly affect the signal intensity versus spectral resolution.

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55 Shutters protect the sample to prevent elongated exposure to light, reducing the effects of photobleaching and photodegradation. A mirror focuses the beam, which is then split. Part of the beam goes straight to the reference detector where the silicon photodiodes output is collected by a current input module. While the rest of the beam hits th e sample and the fluorescence is passed on to the signal detector which uses a photon counting module. It should be noted there are several types of light sources available for excitation s uch as pulsed xenon, high pressure mercury Xe Hg arc, quart tungst en halogen (QTH), lower pressure Hg and Hg Ar lamps, as well as LEDs and laser diodes. Signal Output Lifetime, quantum yield ( Q ) anisotropy or basic emission spectra are used to characterize the process and can be expressed in terms of the emissive rate of the fluorophore ( ) and its rate of nonradiative decay ( nr ) as shown in the equations below. (2 9 ) (2 10 ) As fluorescence is random in nature, only an estimated 37% of the molecule s decay at longer times than the lifetime. Majority emit prior to the lifetime, which is why the quantum yield (Q), the ratio of the number of photons emitted to the number of photons absorbed, is never 100%. Other contributing factors to rapid loss are en vironmental which may lead to thermal loss due to solvent relaxation, quenching, energy transfer, complex formation and excited state reactions 68 These non radiative processes cause a red shift also known as the Stokes shift, in the obtained emission spectra, as the e nergy of emission is lower in comparison to the excitation energy (Figure 2 12) Similar

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56 to previously described spectroscopic methods, emission is recorded as the intensity versus wavelength Another type of measurement, f luorescence anisotropy is used t o provide information about the relative size and shape of molecules or the rigidity of the environment in which there are located. 69 Specifically in this technique, polarized light is applied to a sample where mainly molecules oriented within a range of angles, along the same a xis as the electric field vector of excitation will transition to a higher energy levels. If the environment is rigid, limited mobility, the photons emitted upon relaxation will also be polarized with respect to the molecule. However, if the environment is relatively fluid and the molecule is able to re orient itself, the intensity of the polarized light captured will be significantly reduced. Anisotropy valu es are a ratio of the polarized light to the total light intensity (Equation 2 6 and 2 7). The inten sity ( I ) subscripts (2 11 ) (2 12 ) represent how the excitation (left) and emission (right) polarizers are mounted : V being vertical and H being horizontal. G fac tor sensitivities to unwanted light to improve the signal to noise ratio. The lifetime of this process is called the rotational lifetime ( ) and directly correlated to the tumbling rate of the fluorescent molecule f rom the time of polarized light excitation to the emission of a photon. This relationship is described by the Perrin equation: (2 13 )

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57 where r is anisotropy, r 0 is the anisotropy measured in absence of rotational diffusion, and is the rotational correlation time. Advantages and Disadvantages T he sensitivity of fluorescence techniques pose a major advantage over previously m entioned absorption techniques; its sensitivity is orders of magnitude higher because emi ssion of light is not a common characteristic of substances, leading to low to zero background signals being detected. However, the sensitivity is dependent on the fluorescing substance and the instrument component properties; meaning the fluorophore molar absorptivity, quantum yield and concentration, along with the efficiency of the optical system, play a significant role. This can be monitored by relative signal to noise ratios or by determining limits of detection and quantification of specific fluoroph ores at specific conditions. Other advantages include small sample volumes and rapid analysis times which can reduces experimental cost. Some disadvantages include previously mentioned distortion of excitation and emission spectra due to wavelength depen dent efficiency. Stray light, light that passes through the monochromator in addition to the light of desired wavelength, is a possible source of interference with measured signals and scattering may occur. Naturally, other areas of concern arise in the de tector: over amplification of low signals and a limited range over which the photon counting remains linear. Therefore, finding optimal conditions for experimental measurements is crucial as will be discussed in Chapter 3. All fluorescent experiments perfo rmed and discussed in this dissertation are based on steady state emission and anisotropy.

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58 Electron Paramagnetic Resonance Electron paramagnetic resonance (EPR) is a s pectroscopic technique that utilizes microwaves to study the transition of magnetic dip ole moments of unpaired electron s. These transitions are influenced by the interaction s between electronic magnetic moments and their local environments, with the application of an external magnetic field Upon application of the field, the randomly orient ed magnetic dipole moments align themselves either parallel ( m s = +1/2) or antiparallel ( m s = 1/2) to the field causing a split in the energy states of the spins (Figure 2 16) ; spins that are parallel to the field have a higher energy than those aligned a ntiparallel. The energy differen ce ( ) created between the spin states known as the Zeeman effect, is directly proportional to the resonance frequency ( ) and can be expressed in terms of the applied external field ( B ) where h g is t he spectroscopic g factor, (2 14) and e (or B ) is the Bohr magneton. A more detaile d review of EPR principles can be found in dissertations 70 71 and other published works 72 73 74 75 Although systems containing paramagnetic centers are not co mmon, the development of site directed spin labeling (SDSL) increased the range of systems to which EPR could be applied Particularly when studying bilayer systems, l ipids and membrane associated molecules such as proteins, which have been modified with a nitroxid e spin label, are employed The nitroxide has a persistent radical that is typically protected by neighboring methyl groups, causing it to be stable and unreactive with its environment. The spin of the radical interacts with the additional magnetic field i nduced by the neighboring nitrogen atom nuclei ( I =1) spins that have a net magnetic moment; this phenomenon is known as the hyperfine interaction. This interaction causes further

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59 splitting of energy levels and results in the following three transition stat es: m s m I +1/2, +1 to 1/2, +1; +1 /2, 0 to 1/2, 0; and +1/2, 1 to 1/2, 1 (Figure 2 17) The spectral line shapes seen in EPR represent the transitions that occur and are directly impacted by the mobility of the spin label in their local environment. S pecifically for the work in this dissertation, the line shapes obtained are from lipids labeled at the headgroup (tempo) and acyl chain (5 7 and 12 doxyl) region s (Figure 2 18) These probes were used to evaluate the accessibility of paramagnetic reage nts in bilayer systems upon surfactant peptide assimilation using powe r saturation continuous wave (CW ) EPR. Pow er saturation CW EPR relies upon the Heisenberg exchange interaction between the spins of the spin label and the paramagnetic reagent; oxygen ( O 2 ) and nickel (II) acetylacetonate hydrate ( NiAA ) are the paramagnetic species utilized the work described in this dissertation When the paramagnetic reagent physically collides with the spin label, it leads to faster relaxation rates. Given the polarity gradient along lipid bilayers, the relaxation effects become a direct measurement of the solvent accessibility to the spin labels. Therefore with peptide incorporation, changes in the accessibility can be used indirectly to measure the relative depth para meter within the lipid environment. Steric hindrance, diffusions constants, and concentrations along the bilayer are the other factors that impact the rate of collisions. Experimental details are described in chapter six. Timescales of Lipid M otions To characterize the effects of surfactant peptides on lipid bilayer dynamics and fluidity, the type of molecular interaction and the timescale of lipid motions must be evaluated with an appropriate technique (Figure 2 19) The f luorescence emissions and aniso tropy techniques previously described are used to monitor intermolecular

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60 interactions and motions in the nanosecond regime; and power saturation CW EPR is used for intramolecular interactions within the same timescale.

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61 Figure 2 1 Thin layer chrom atogram of the negative control LPC DPPC:POPG and POPC:POPG mixtures, visualized by staining with iodine vapour E ach phospholipid component is circled and labeled (a) LPC (b) DPPC (c) POPC and (d) POPG.

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62 Figure 2 2 Electromagnetic spectrum. Figu re 2 3 Plane polarized electromagnetic radiation propagating along x axis ( http://micro.magnet.fsu.edu/primer/java/wavebasics/ )

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63 Figure 2 4 Molecular energy level diagram showing the transition from the ground state (S 0 ) to first (S 1 ) level excited state, internal conversion, vibrational relaxation and fluorescence: the emission of energy and return to S 0 ( modified from the Jablonski diagram ). Figure 2 5 Correlation diagram of th e energy states of hydrogen atoms before and after formation of diatomic hydrogen (H 2 ): lowest energy state is the bonding

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64 Figure 2 6 Electronic transitions and respective energy levels. Table 2 1 Molecular moieties likely to absorb light in the UV Vis region Chromopho re Example Excitation max nm Solvent C=C Ethene __ > 171 15,000 hexane 1 Hexyne __ > 180 10,000 hexane C=O Ethanal n __ > __ > 290 180 15 10,000 hexane hexane N=O Nitromethane n __ > __ > 275 200 17 5,000 ethan ol ethanol C X X=Br X=I Methyl bromide Methyl Iodide n __ > n __ > 205 255 200 360 hexane hexane

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65 Figure 2 7 UV Vis Spectrophotometer schematic. Figure 2 8 Absorption spectrum of cuvettes made from different materials (conta ining water) and possible wavelengths of interference.

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66 Figure 2 9 The formation of elliptically polarized light from left (E L ) and right (E R )

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67 Figure 2 10 Graphical representation of polarized light shifted out of phase to produce circularly polarized light Figure 2 11 Peptide bond transitions.

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68 Figure 2 12 Circular dichroism standard protein structure spectra ( ht tp://www.proteinchemist.com/cd/cdspec.html ) Figure 2 13 Absorption and fluorescence emission spectra.

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69 Figure 2 14 Block diagram showing general schematic of spectrofluorometer. Figure 2 15 A schematic of the spectrofluorometer components, as displayed in the Fluoromax 3 manual. 1 Xenon arc lamp and lamp housing, 1a Xenon lamp power supply 1b Xenon flash lamp (does not apply to instrument used in proposal) 2 Excitation monochromator, 3 Sample compartment 4 Emission monochroma tor, 5 Single detector (photomultiplier tube and housing), 6 Reference detector (photodiode and current acquisition module), 7 Instrument controller

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70 Figure 2 16. Energy level diagram for a free electron in an applied magnetic field showing the Zeeman i nteraction. Figure 2 17. Energy diagram showing the hyperfine splitting of an electron with m s = 1/2 coupled to a nucleus with m I = 1.

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71 Figure 2 18. EPR spectral line shapes for labeled at the headgroup (tempo) and acyl chain (5 7 and 12 doxyl ) regions of a lipid. Figure 2 19. Timescales of different types of lipid motions in a lipid bilayer model (adopted from Adam Smith).

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72 CHAPTER 3 EXPERIMENTAL OPTIMIZATION Introduction Intrinsic and extrinsic fluorescent probes were selected based on their chemical structures and predicted localization within the following liposomes of interest: DPPC:POPG and POPC:POPG. 1 Palmitoyl 2 (pyrene 1 yl)decanoyl sn glycero 3 phosphocholine (PPDPC) and diphenylhexatriene (DPH) were utilized to interrogate the hydrophobic core, trimethylammonium diphenylhexatriene (TMA DPH) for the plateau region located at position 2 to 10 on the acyl chain 76 and rhodamine PE for the bilayer surface/headgroup area ( Fi gure 3 1). Each lipid system were characterized using fluorescence emission and anisotropy spectroscopy and DSC, to determine optimal experimental parameters and to monitored for fluorescent probe effects on bilayer dynamics and fluidity prior to peptid e incorporation. Initial concentration dependence studies were used to select a probe concentration that would maximize the differences seen in steady state emissions with minimal perturbation to the local environment. Anisotropy measurements were able to track variances in gel and amorphous regions of the liposomes and yielded thermophysical information 77 that were comparable to DSC, phase t ransition temperatures (T m ). Vesicle Preparation Mixtures of the most abundant lipids (DPPC, POPC, and POPG) 15 7 in the surfactant were used to prepare liposomes (or lipid vesicles) as a synthetic representation of the subphase, which is located directly under the monolayer of the air fluid interface in alveoli. As DPPC is known to be the predominant phospholipid at the surface where oxygen absorption occurs, and POPG is significant for surfactant

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73 spreading, a molar ratio of 4:1 DPPC:POPG was utilized as the model system with the hypothesis that peptide effects would be more prevalent in this system. A 4:1 POPC:POPG mixture is used as a standard for comparison. Prior to liposome preparation, the structural integrity and concentration of each lipid stock solution was verified by TLC and m alachite g reen p hosphate colorimetric assay (BioAssay Systems, Hayward, CA) Lipid Structure and Concentration PPDPC, DPH and TMA DPH were purchased from Sigma Aldrich (St. Louis, MO ). Rhodamine PE, DPPC, POPC, and POPG were purchased as chloroform based solutions from Avanti Polar Lipids (Alabaster, AL). The structural integrity of the lipids was verified by TLC analysis on silica plates. Because of the amphipathic nature of the phos pholipids, a solvent mixture of volume ratios 65:25:4 chloroform/methanol/water was used as the mobile phase. For each analysis, both positive and negative controls were run. Specifically, the positive being a stable phospholipid that was stored in a teflo n sealed vial that had been purged with nitrogen gas, and determined to be structurally viable; the negative being a lyso phosphatidylcholine (LPC), a standard degradation product of phospholipids. The stock phospholipid stock solutions were allowed to equ ilibrate to room temperature prior to opening the vials to reduce the rate of volatilization due to suspension in chloroform. Standard procedures were followed. The malachite green phosphate assay is based on the quantification of the complex formed betw een malachite green, molybdate and free phosphates. A standard curve (Figure 3 2) is generated from a series of standard samples containing known concentrations of aliquoted inorganic phosphate solution. To generate free phosphates from the phospholipids, exposure to harsh acid (8.9 N sulfuric acid) and elevated

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74 temperature (220 C) conditions is necessary. After which the solutions are cooled, neutralized with sodium hydroxide (1 M), vortexed and subsequently mixed with the assay solution. These solutions are incubated at room temperature for at least 10 minutes to ensure completion of reaction, which yields color formation, and then absorbance readings are taken at 650 nm. The values obtained are then used to determine more accurate concentrations of the p urchased phospholipids This assay is performed periodically to track possible changes in individual lipid stock solution concentrations resulting from solvent evaporation. Liposome Formation A series of steps are followed to enable proper formation of l arge unilamellar vesicles (LUVs). The first step was to compute the volumes of each individual stock lipid solutions, concentration was determined by phosphate assay, needed to make mole ratio mixtures of 4:1 DPPC:POPG and 4:1 POPC:POPG ; both were aliquote d into 1.5 mL amber glass vials A stream of nitrogen gas was blown over the mixtures for solvent removal. A specific volume of chloroform was added to the each lipid film pair, to yield a total lipid solution concentration of 1 mM. The mixture was quickly sealed with polytetrafluorothethylene (PTFE) lined caps and parafilmed to prevent unwanted evaporation, followed by vortex mixing. This solution becomes our lipid stock to be used in preparation for vesicles with the various probes of interest. After addi tion of the probe, the samples are dried with a stream of nitrogen gas, followed by placement in a vacuum dessicator overnight. For samples containing peptide suspended in methanol, they are reconstituted in cyclohexane, flash frozen in liquid nitrogen and quickly placed on the lyophilizer overnight. Placement in a reduced pressure environment ensures removal residual organic solvent; producing a dry lipid film/cake of liquid crystalline bilayers.

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75 Following the drying process, hydration in a 5 mM 2 [4 (2 hydroxyethyl)piperazin 1 yl]ethanesulfonic acid (HEPES) buffer solution ( 0.1 mM EDTA, 100 mM NaCl, 0.02% NaN 3 (w/v) pH 7.4 ) at a temperature (45 C) above the lipid phase transition temperature (T m ) occurs with periodic vortexing. Samples are maintained at this temperature in an incubator oven and are subjected to at least 5 freeze thaw cycles in liquid nitrogen and water respectively to yield multilamellar vesicles (MLVs). Repeated freeze thaw cycles have been shown to reduce the presence of non equili brium solute distributions caused by osmotic imbalances between the interior and exterior of vesicles. 78 79 80 After a stable and homogen ous suspension is created, sonication or extrusion can be performed to generate vesicles of specific and uniform sizes, which can be verified with dynamic light scattering (DLS). In all experiments, we have used lipid extrusion for the formation of LUVs. Extrusion is a technique where mechanical force is used to push lipid suspensions through polycarbonate (PC) filter membranes to produce LUVs of comparable size to that of the PC pores. A 100 nm multiporous PC membrane filter was mounted into an extrusion cell (purchased from Avanti Polar Lipids), and placed on a holder/heating block (Figure 3 3) which was been pre equilibrated on a hotplate for 45 minutes at 50 C. The MLV dispersion was loaded into a 1 mL glass syringe, placed in one end of extruder cell; an empty 1 mL glass syringe was mounted on the opposite side. The MLV dispersion was mechanically pushed through the PC membrane pores, with only LUVs dispersions of a specific size range being collected in the other syringe. This process was repeated 31 times to maximize the number of LUVs produced and ensure a narrow size distribution. 81 Immediately after this step is completed, the

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76 collected LU V dispersion is loaded into a 4 mm quartz cuvette, placed in the fluorometer, and data are collected. Subsequent LUV dispersions that were collected after extrusion, were placed into a 45 C incubator until data collection for earlier samples were complete d. All sample data were collected soon after extrusion because it is know n that with long delays suspensions begin to lose size homogeneity due to aggregation. Fluorescent Probes P yrene Pyrene is a polycyclic aromatic hydrocarbon (PAH) used in various sc ientific stud ies due to its exhibition of the following beneficial properties: its long singlet lifetime, its sensitivity to environment polarity and its ability to act as an energy donor or acceptor in non radiative energy transfer 82 83 84 85 O ne of its most beneficial properties in studying biological membrane dynamics is the ability to form excited state dimers also known as excimers An excimer is a dimer that is associated in a n excited electronic state and is dissociative in its ground electronic state 86 Analogous to some of the spectroscopic properties re corded with noble gases, the electrons at the ground state undergo transitions to both sing let and triplet excited states as can be described in the following four step process (I IV), the first three being identified by Foster and Kasper, 87 where 1 M* => 1 M + M (I) 1 M* + 1 M <=> 1 D* (II) 1 D* => 1 M + 1 M + D (III) 3 M* + 1 M <=> 3 D* (IV) 1 M* and 1 M are the monomeric singlet states at excitation and ground levels, 1 D* is the excited st ate dimer, 3 M* and 3 D* are the triplet excited states of the monomer and

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77 dimer. These processes lead to a series of vibronic bands based on distinct radiative transitions from the excited singlet states to different vibrational levels of its ground state 88 Therefore solute concentration and polarity of the solvent will modify the fluorescence emissi on spectra ; self quenching occurs at high concentratio ns. Depending on the local environment and polarity, a broad structureless band may be present. The are two types of molecular interactions said to govern excimer formation: e xciton resonance and charge resonance 89 Exciton resonance is due to dipole dipole interaction between excited and ground state molecules, wh ereas charge resonance is a result of Coulombic interactions between positive and negative molecular ion states. 82 Alt hough both models factor in the importance of separation distance between molecules and overall orientation, neither factored in the need for orbital overlap between molecules 90 Over the years, there have been multiple debates on whether excimer formation is a resonance dipole or a charge transfer based interaction. Depending on the sy stem being studied, experimental results have been shown to support both mechanisms. In general, the rate of excimer formation is directly affected by temperature and solvent viscosity 86 91 Considering its sensitivity t o the surrounding environment, when tethering a pyrene to the acyl chain of a phospholipid and integrating into a biological membrane, differences from the standard organic solution spectra of pyrene should be expected. The use of pyrene labeled lipid pro bes is growing as a tool to understand deviations from normal characteristic behavior 92 93 94 and for exploring membrane dynamics 95 96 56 Some studies used the rate of excimer formation by pyrene based fatty

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78 acids in membranes to gain information on the dynamic properties of artificial and natural fluid lipid membranes. Through these experiments, lateral diffusion coefficients were also extracted Determination of transv ersal mobility, the exchange of lipids between layers of a djacent bilayers or between bilayers of different vesicles, has been possible with this technique. With limited local perturbations to the native environment, various intermolecular interactions of neighboring lipids can also be detected 56 97 98 Contradictory studies have also shown the mechanism of excimer formation within membranes may not be solely diffusion controlled as aggregates may be present due to the biphasic nature of lipid bilayer environmen t s ; 92 though these studies stated slow diffusing monomeric pyrenes were present. Overall pyrene labeled phospholipids within bilayers have been useful in monitoring p hospholipid phase transitions, a cyl chain interdigitation, acyl chain alignment, fluidity, lateral diffusion, etc DPH and TMA DPH As mentioned previously, fluorescence anisotropy has been utilized in studying membrane fluidity, particularly because the polarization intensities are dire ctly correlated to environment viscosity. Measuring the anisotropy of extrinsic probes such as diphenyl hexatriene (DPH) and N,N,N Trimethyl 4 6(6 phenyl 1,3,5 hexatrien 1 yl)phenyl ammonium (TMA DPH), which have been incorporated into biological membranes is a within the local vicinity. Particularly DPH is commonly used because it is know n to partition favorably into membranes and does not appear to bind to proteins which may be present in the bilayer How ever, as shown in Figure 3 1, DPH may adapt two orientations within a bilayer leading to complications in data analysis. The modified TMA DPH became of interest in that the positive charge on the ammonium group could electrostatically

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79 interact with the pol ar headgroup s of phospholipid bilayers and lead to alignment within the plateau region Rhodamine PE Rhodamine PE is another intrinsic probe that is typically used in fluorescence resonance energy transfer (FRET) studies designed to monitor membrane fusio ns events. Due to the bulky nature of the tethered chromop hore, other studies evaluated the perturbation effects of the fluorescent probe on bilayer structural integrity 59 The conclusions of this work showed that within specific concentration ranges, organization within bilayer systems could be interrogated and other parameters to describe the local environment could be extracted (ex. wobbling diffusion constants). Alternatively to steady state anisotropy time resolved measurements could be made where the recorded experimental data is based on Equation 3 3 ; anisotropy decay measur ements Due to direct attachment of the rhodamine to a phospholipid, the motional freedom is (3 3) (3 4) (3 5) restricted, for the non zero infinite time anisotropy. From this data, the time constant ( HR ) and cone a ngle ( ) can be extracted from E quations 3 4 and 3 5 These values will then be used to calculate the wobbling diffusion constant ( D w ). This constant will prove very significant in monitor ing changes in rotational motion should the chromophore interact (3 6)

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80 between t w o different lipid phases, or with modifications in the local environment dynamics upon peptide binding. Despite the complexity of this system, surface activity information could potentially be used to verify phase separations that may occur in our lipids systems which is crucial for mapping the trafficking mechanisms of the surfactant. Experimental Parameters and Results PPDPC Following extrusion, the 4:1 POPC:POPG and 4:1 DPPC:POPG samples containing PPDPC were allowed to equilib rate in a temperature controlled sample cell of the FluoroMax 3 fluorometer for 5 minutes at 45 C. The excitation wavelength was set at 344 nm, while emission data was collected from 360 550 nm to cover the multiple vibronic bands typically displayed wi th pyrene emission. All plotted emission spectra comprised three set of scans averaged during data processing and data were reproduced three times The excitation and emission band passes were set to 5 nm, and the excitation and emission polarizers were s et to 90 and 0 respectively Measurements were made using a 4 mm light path quartz cuvette. Background subtractions, using emission spectra of buffer, are done on all the spectra. As mentioned previously, to determine optimum probe concentration that ca uses the least amount of perturbation while maximiz ing signal output for observation of changes in bilayer dynamics and fluidity, we varied the concentration of the probes and monitored their fluorescence emission. In the PPDPC studies, the probe concent ration was varied from 1 to 10 mol% in our 4:1 POPC:POPG and 4:1 DPPC:POPG liposomes and the steady state emission spectra of our s amples were recorded (Figure 3 4 ). Both systems exhibited classic pyrene spect ra with a reduced number of vibronic peaks in comparison to pyrene

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81 suspended in organic solutions, due to the more rigid nature of the lipid bilayer environment. For all studies, peaks at 376 and 395 nm are referred to as the monomer peaks, wh ereas the broad peak at ~480 nm is the excimer peak; peaks at 395 nm and 480 nm are referred to as the monomer ( I m ) and excimer ( I e ) peak respectively After normalizing the fluorescence intensity of the monomer peak at 376 nm to one the ratio of I e to I m was substantial differences in the pyrene rate of excime r formation were visible in the DPPC:POPG mixture r elative to POPC:POPG (Figure 3 5 ). For both lipid systems, t he monomeric emissions stayed relatively constant and overlapped while the excimer emissions increased linearly with probe concentration (Figure 3 5a ) The steeper I e /I m slope in the DPPC:POPG system (Figure 3 5b) was attributed to the high content of the fully saturated DPPC lipid leading to tighter packing ; this causes an increase in ordering and less fluidity. For all subsequent experiments, we chose 3 mol% PPDPC for minimal perturbation of bilayer and used the I e /I m ratio is used to evaluate changes. To monitor phase behaviours of the lipid systems with the probe, we varied the temperature from 25 to 45 C in +2 C increments (Figure 3 6 a). T he I e /I m of the POPC:POPG system stayed relatively constant with a slight increase from 0.1600.001 to 0.2060.003 in emissions ; this result is expected given the 2 C T m of both monounsaturated lipids which is well below room temperature. The DPPC rich system underwent a phase transition within the range of 33 to 37 C shown by the ~ 0.3 decrease in I e /I m At lower temperatures, the error bars are larger as may be due to the amorphous and gel properties of the mixture; with increasing temperature the sam ple becomes more homogeneous, as can be seen with less deviation in the I e /I m DSC

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82 measurements (Figure 3 6 b) of the DPPC:POPG liposomes containing 3 mol% PPDPC corroborate the transition seen in temperature studies, provides T m 34.7 C. DPH Similar to the methodology described for the PPDPC section, samples of 4:1 POPC:POPG and 4:1 DPPC:POPG were made containing probe concentration s from 0.1 to 5 mol% DPH. The excitation wavelength was set at 360 nm and the single point anisotropy value was measure d at 432 nm Peak maxima were determined by recording the steady state emissions spectra at each probe concentrat i on All other experimental parameters were the same as previously discusse d In Figure 3 7a a distinct separation in the anisotropy values measur ed in POPC:POPG vesicle mixtures (~0.23) in comparison to the DPPC:POPG vesicles (~0.13) is observed. T h e data show that DPH is less mobile in the POPC:POPG bilayer system suggest ing that it preferentially orients itself within specific regions of the bila yer leaflet as drawn in Figure 3 1. Specifically in the DPPC:POPG system, lipid packing and ordering may prevent integration of the probe into the acyl chain region essentially forcing it to rotate in between the two layers (bilayer leaflets) of the hydrop hobic core ; and the fluid nature of the POPC:POPG vesicles would enable penetration and alignment of the probe along the acyl chains, which may lead to ordering of bilayer. The intercalation of the probe between acyl chains increases the anisotropy value b ecause the ; reducing reorientation of the fluorophore and increasing the detected polarized emissions signal. Based upon the results of these experiments, we selected a 0.5 mol% DPH concentration for minimal perturbation to local environment, to monitor phase transitions with temperature increases as previously described. At lower temperatures,

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83 t he anisotropy values of DPH in the DPPC:POPG vesicles (Figure 3 7b) are at higher values (~0.33) than sh own in the concentration dependence studies ( ~0.13 ) suggesting the liposomes are in gel phase, which would limit rotational diffusion of the probe. A s the temperature increases, a decrease in the measured polarized light begins around 33 C which is indic ative of the transition to the fluid phase. The POPC:POPG system shows a slight decrease in anisotropy from ~0.27 to 0.24 with increasing temperature Although the POPC:POPG does not undergo a phase transition, the slightly higher anisotropy value is belie ved to be an effect of the probe localization in between acyl chains and reduced motion at lower temperatures TMA DPH TMA DPH has a lower quantum yield than DPH due to the ammonium modification. For this reason, concentration variations were at higher r anges 1 to 10 mol% probe The excitation and emission wavelengths are 360 nm and 432 nm respectively, same as the DPH settings. The anisotropy values are similar for both lipid systems in the 2 to 8 mol% TMA DPH range (Figure 3 8) This finding is expecte d because the positively charged ammonium group would likely interact with the headgroup phosphates on the acidic (POPG) and zwitterionic (POPC and DPPC) lipids causing the probe to align at the plateau region of both lipid systems For the temperature st udies, we selected 2 mol% to minimize local perturbations A decrease in anisotropy is observed from 33 to 35 C (Figure 3 9a) in our DPPC:POPG liposomes, indicating a phase transition occurred DSC data confirmed this value with a measured T m of 34.4 C ( Figure 3 9 b)

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84 Rhodamine PE The relatively low probe concentration range (0. 0 1 to 0.4 mol%) was selected based on recommended literature values due to the bulky nature of the rhodamine headgroup ; it is known to perturb liposome organization at higher conc entrations. 59 At low probe concentrations (Figure 3 10a) the anisotropy valu es of ~0.3 depict a rigid environ ment and decreases as the probe concentration increases This result is comparable to previous work which described less interaction between chromophores at low concentrations ; and how the polar nature and flexibility of th e headgroup enabled concentration increases, a decrease is observed in anisotropy values, which is indicative of bilayer environment perturbation. A 0.05 mol% rhodamine PE concentration was used for the temperature experiments. The DPPC values overlap with the concentration dependence studies, and no visible phase transition is observed which is expected due to the location of the chromophore. However, the POPC system appe ars to exhibit significantly higher anisotropy values (Figure 3 10 b) Discussion and Conclusions We selected both intrinsic and extrinsic fluorescent probes and monitored their effects on the 4:1 DPPC:POPG and 4:1 POPC:POPG bilayer systems. T he I e /I m rat ios computed from PPDPC steady sta te emissions spectra were useful in monitoring the relative phase transition of our 80 mol% saturated lipid system, and for selection of a probe concentration that would cause minimal perturbations to the local environment while maximizing signal output for peptide experiments described in later chapters DPH yielded unexpected results which brought into consideration the packing dynamics and pr eferential orientation of extrinsic probes within bilayer systems This probe w as not

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85 selected for future work, as the differences in localization would not produce comparable results to monitor effects of peptide on the different lipid bilayer systems. PPDPC, DPH and TMA DPH were useful for phase transi tion temperature determinatio ns. T he observed changes in I e /I m versus temperature with PPDPC experiments, and the anisotropy ve rsus temperature measurements with TMA DPH were comparable to the experimental T m values acquired with DSC These results suggest that integration of the fluo rescent pro bes 3 mol% PPDPC and 2 mol% TMA DPH, does not have major effects on the physical properties of our bilayer systems. The results of rhodamine PE fluorescence anisotropy experiments in bilayers were similar to published work. At low probe concent ration, 0.05 mol%, unusually high anisotropy values were detected in the POPC:POPG system. It can be hypothesized that the inversion of the bilayers may have occurred because the headgroup interactions are stable in comparison to the fluid and weak van der Waals forces in acyl chain region of the monounsaturated bilayers, causing lipid phase changes and inevitably scattering the polarized light The rhodamine PE probe proves a more complicated system for analysis it can be used to monitor the peptide induc ed transformation to novel phases as observed with NMR studies by the Long research group (University of Florida, Gainesville, FL).

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86 Figure 3 1. Fl u o rescent probes used throughout this dissertation Figure 3 2. A) Malachite green phosphate assay standard curve used to determine phospholipid concentrat ion prior to sample preparation: the error bars represent the deviation from the mean value of triplicate runs. B) A picture of cuvettes that contained increasing concentrations of phosphate standard

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87 Figure 3 3 Mini extruder set up with holder/heating block and mounted syringes (Avanti Polar Lipids, Alabaster, AL).

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88 Figure 3 4 Emission spectra show c oncentration dependence effects on the excimer (I e ) and monomer (I m ) emissions of varied mol % PPDPC in A) 4:1 POPC:POPG and B) 4:1 DPPC:POPG liposomes. Total lipid concentrations were 25 M in a 5 mM HEPES buffer solution (pH 7.4). Data were collected in triplicate using an excitation wavelength of 344 nm at 45 C The spectra collected at each m ol% PPDPC was averaged and then normalized with respect to the first peak at 376 nm

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89 Figure 3 5 Normalized fluorescence emission intensity of 1 to 10 mol% PPDPC in 4:1 DPPC:POPG (red circles ) and 4:1 POPC:POPG (black triangles ) A) The results of monomer emission intensity (I m ) is at a steady value below 1; the excimer emission intensity values (I e ) increase linearly relative to probe concentration. B) Shows I e /I m values with increasing probe concentration. 7.4). Data were collected in triplicate at 45 C with an excitation wavelength of 344 nm Error bars represent the standard deviation of measurements from three different samples pre pared from the same stock lipid mixture. Figure 3 6 Thermotropic p hase transitions of liposomes containing 3 mol% PPDPC A) Results show the excimer to monomer (I e /I m ) values in 4:1 DPPC:POPG (red circles) and 4:1 POPC:POPG (black triangles) liposomes with increasing temperature from 25 to 45 C (+ 2 C increments). Total lipid concentrations triplicate ; error bars represent the standard deviation of measurements from three diff erent samples. B) DSC thermogram of 4:1 DPPC:POPG liposomes (1 mM) containing 3 mol% PPDPC with a T m of 35 C ; the scan rate was 1 C per minute

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90 Figure 3 7 Fluorescence anisotropy results of 4:1 DPPC:POPG ( red circles ) and 4:1 POPC:POPG (black tria ngles ) with A) increasing DPH concentration: 0.01 to 5 mol%. solution (pH 7.4). Data were collected in triplicate at 45 C with an excitation wavelength of 360 nm and emission wavelength of 432 nm B) Anistropy values of liposomes containing 0.05 mol% DPH with increasing temperature from 25 to 45 C (+ 2 C increments). Error bars represent the standard deviation of measurements from three different samples prepared from the same stock lipid mixture.

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91 Figure 3 8 Fluorescence anisotropy results o f 4:1 DPPC:POPG (red circles ) an d 4:1 POPC:POPG (black triangles) with increasing TMA DPH concentration: 1 to 10 mol%. solution (pH 7.4). Data were collected in triplicate at 45 C with an excitation wavelength of 360 nm and emission wavelength of 432 nm. Error bars represent the standard deviation of measuremen ts from three different samples prepared from the same stock lipid mixture. Figure 3 9 Thermotropic phase transitions of liposomes containing 2 mol% TMA DPH. A) Results obtained from fluorescence anisotropy measurements 4:1 DPPC:POPG (red circles) and 4:1 POPC:POPG (black triangles) liposomes with increasing temperature from 25 to 45 C (+ 2 C increments). Total lipid were collected in triplicate; error bars represent the standard deviation of measurements from three dif ferent samples. B) DSC thermogram of 4:1 DPPC:POPG liposomes (1 mM) containing 2 mol% TMA DPH with a T m of 34 C ; the scan rate was 1 C per minute

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92 Figure 3 10 Fluorescence anisotropy results of 4:1 DPPC:POPG (red circles ) and 4:1 POPC:POPG (black triangles ) with A) increasing rhodamine PE concentration : 0.01 to 0.4 mM HEPES buffer solution (pH 7.4). Data were collected in triplicate at 45 C with an excitation wavelength of 557 n m and emission wavelength of 587 nm. B) Anisotropy measurements ob tained from liposomes containing 0.05 mol% rhodamine PE with increasing temperature from 25 to 45 C (+ 2 C increments). Error bars represent the standard deviation of measurements from three different samples prepared from the same stock lipid mixture.

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93 CHAPTER 4 EFFECTS OF pH ON KL 4 PEPTIDE STRUCTURE AT MEMBRANE INTERFACE Introduction Biological membranes are generally known as boundaries that separate the cytoplasm and cellular organelles from the exterior of the cell 99 However, providing a barrier to the solute flux between aqueous compartments is not the only crucial part of their function 100 Ion transport, surface adsorption or partitioning of molecules into bilayer interior, and the orientation of membr ane proteins, are examples of relevant biochemical processes, which depend on interactions between the molecular species and lipid bilayers. These types of interactions also play a significant role in determining the effective binding of therapeutic drugs and peptides. The interface at the biological membrane is a complex system that is governed by precise molecular and ionic interactions within the local environment. Therefore to further understand and model these biological systems, various mechanical and electrical properties of membranes 101 must be considered. Surface Potential As mentioned previously, when lipids are solubilized in aqueous media, they self assemble into various structures; the specific polymorphism generated is based on the hydrophobic effect and sh ape hypothesis. Throughout this dissertation, lipid vesicles (liposomes) were utilized to mimic the subphase extracellular environment of the alveoli. However, to understand the effects of synthetic surfactant peptides on bilayer dynamics and fluidity, it is important to consider the local environment into which they are integrated.

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94 Bilayers form to prevent hydrophobic carbon tails (acyl chains) from coming in contact with polar molecules increasing the entropic disorder of solvating water molecules. This separation of water and hydrocarbon creates an energy barrier to charged molecules, and effectively reduces membrane permeation. 102 Most biological membranes have an overall negat ively charged surface due to the presence of anionic phospholipids (ex. POPG), typically making up about 10 to 20 mol% of the bilayer. In some cases, other membrane components may contribute to the negatively charged surface. Naturally, counter ions presen t in the surrounding fluid phase will neutralize this charged surface but are neither fixed nor permanently bound, to the membrane surface. Therefore, the distributions of these charged counter ions are dependent on the overall surface charge density a nd t he surface potential creating an electrical double layer. The electrical double layer is a diffusion controlled process that describes the charge gradient created by membrane surface charge density and the ions from bulk solutions 103 This gradient creates an electric potential at the membrane surface, which is dependent on the concentration and valence charge of ions in the aqueous env ironment. In the early 1900 s, the Guoy Chapman theory was developed as a means to describe electric potential energy as a function of distance from the membrane surface 100 Based on the complexity of ion mobility, valence charge, and net membrane surface charge per area, the following four main assumptions wer e made: (1) the surface charge is uniformly distributed across the surface therefore can not be treated as individual points (2) ions are simple point charges irrespective of the individual size (3) repulsion of ions moving across the interface are ignored (4) the dielectric constant is the same in bulk solutions as at the membrane interface. Though this theory was

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95 shown in the 1920 s used ionic s ize to compute the maximum number of counterions th at can interact with the surface at any given point in time This l ocal ion concentration is modeled by the Boltzmann equation where C(X) and (X) are the ion concentration and electric (4 1) potential at a distance X relative to the surface. C( is the bulk concentration Z is the ion valence F R is the gas const ant and T is the temperature of the system. T he effects of charged membrane surfaces on the gradient of ions concentrated wi thin proximity of that surface have proven that the Gouy Chapman Stern theory is an effective model for most systems However, a det ailed review described the evaluation of light d ependent reactions at thylakoid membrane interfaces in plant cells, and determined there are other significant factors not accounted for in the theory 104 One challenged the planar view of the membrane surface, consider ing integral protein s may protrude from the surface and extrinsic proteins may be absorbed at them. Another main factor relates to the changes in pK values of membrane surface s because the activity coefficients of ionisable species differ depending on their localization in th e bulk media versus the Helmholtz layer 104 105 Surface pH Each biochemical process requires a specific set of condition s for proper function. Therefore each parameter must be considered when attempting to extract information about the local environment. T emperature, ionic strength, and bulk concentration all affect the surface potential and inadvertently the way ions and

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96 m olecules interact at the membrane surface Particularly with a charge gradient, which is directly influenced by ionic strength, ionisable species tend to deviate from ideal behaviors at the membrane surface versus the bulk medium ; membran es typically have phospholipids that have ionisable species at their headgroup. Equation 4 2 is used to model the effects varying activity coefficients can have on the dissociation constant of (4 2) i onisable species; where pK obsd is the observed pK a pK int is the intrinsic pK a A and HA are the activity coefficients of the conjugate base and acid respectively 105 As described in a study to extract the pK values for PS and PE in PC bilayers 106 the intrinsic pK a is defined as the surface pH at which half the population is charged, while the apparent pK a is the bulk pH at which half the population is charged. T heir relationship is shown by the equations below. (4 3) (4 4) Though pH 7.4 is considered a standard condition in biological systems, ther e are significant effects on me mbrane surface pH as ions (ex. s alts) can decrease the electric potential at the surface 107 and in turn modify the degree of ionization of phospholipid headgroups. Specif ically at the membrane int erface, acidic phospholipids and proteins with charged residues can contribute to the surface charges. T o evaluate the effects of pH on the KL 4 peptide (SP B 59 80 mimic) secondary structure at the membrane interface CD spectroscop y is utilized

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97 Material and Methods Synthesis of KL 4 The peptide was previously made through automated solid phase synthesis on a Wang resin (ABI 430, ICBR, University of Florida). It was then cleaved from the resin with 90% TFA/5% triisopropyl silane/5 % water and ether precipitated. The crude product was purified by reverse phase high performance liquid chromatography using acetonitrile/water gradient, and purity was verified by mass spectrometry. Weighing 10 mg of dried peptide and dissolving in 3 mL H PLC grade methanol made a stock solution A calibration curve generated from measurements of previous by amino acid analysis (Molecular Structure Facility, University of California, Davis, CA ) samples was used to determine the peptide concentration. UV spe ctroscopic measurements were done in triplicate at 214 nm where amide backbones have a high absorbance and a final concentration of 5.4 mM was calculated An aliquot of the 5.4 mM peptide solution was used to make a 320 M stock for all samples prepared. Preparation of L iposome S amples POPC, DPPC, POPG were purchased as chloroform solutions (Avanti Polar lipids, Alabaster, AL) and concentrations were verified by phosphate analysis (Bioassay Systems, Hayward, CA). Lipid stocks were mixed in chloroform to o btain molar ratios of 4:1 POPC:POPG and 4:1 DPPC:POPG and aliquoted based on predetermined experimental concentration. The peptide methanol solution was mixed with lipids to achieve approximately 2 mol% (40 M) in a 2 mM final lipid concentration. The samples were dried under a stream of nitrogen gas in a 42 45 C water bath; the films were then reconstituted in cyclohexane, flash frozen in liquid nitrogen, and lyophilized overnight to remove residual solve nt.

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98 CD E xperiments A series of buffers were prepared at the following pH values: 6.4, 6.7, 7.0, 7.3, 7.6 and 7.9. Sodium phosphate monobasic monohydrate and Sodium phosphate dibasic anhydrase (Fisher Scientific Waltham, MA) were used to make phosphate b uffers with the aforementioned pH values t o hydrate one group of samples. At elevated temperatures, the acid dissociation of phosphates is known to increase, so a second set of buffers were made with Bis Tris propane (Sigma Aldrich, St. Louis, MO) at the s ame pHs Stock buffer solutions of 10 mM were made with 140 mM sodium chloride. These were used to hydrate the pre aliquoted dried lipid peptide films at 45 C with vortex mixing. Then the suspensions underwent 5 freeze thaw cycles and were left in an incu bator overnight set at 45 C. The MLV dispersions were then extruded through a 100 nm polycarbonate membrane (Millipore, Bedford, MA) 31 times to separate into LUVs and immediately underwent CD analysis All experiments discussed in this section, were per formed on an AVIV Model 202 (Lakewood, NJ) at 45 C. A step scan method was used with a start and end wavelength of 260 nm and 200 nm respectively, a 1 nm step size, averaging time of 4 seconds and 16 scans. Stock buffer solutions were used for background subtraction. All measurements were done with a 1 mm quarts cuvette. Results and Discussion As mentioned in the introduction, many biochemical processes rely on membrane surface charges for proper function 108 and with approximately 10 to 20 mol% of phospholipids present at bilayer membranes having charged headgroup s this induces a surface potential; directly affecting the immediate environment at the interface. 109 The balance between counterions present in the external environment and

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99 the presence of other charged moieties such as the lysine residues of KL 4 peptide, drastically affect s the local pK a values at the interface. These changes in electrostatic 110 dynamics can impact the overall vesicle morphology and provide different physical constraints on membrane bound peptides such as conformational changes. 111 112 113 The figures in this chapter show CD spectra of liposomes prepared with peptide. The first set of experiments were performed in 10 mM NaH 2 PO 4 H 2 O /Na 2 HPO 4 with 140 mM NaCl buffer at 45 C (Figur e 4 1 ); t he approximate pH valu es of the buffering environment are given in the legend Due to chemical properties of the buffer, there is an increase in acid dissociation at temperatures above 20 C, which directly affects the pH. The pK a at temperatures above 20 C is approximated to change at a rate of 0.03 pK a units per C, therefore lowering the each pH value by ~0.6 units; for example the actual pH of the pH 6.4 buffer would be 5.8. In the 4:1 DPPC:POPG system, characteristic alpha helical minima are seen at ~222 nm and 208 nm fo r pH 7. 3, 7.6 and 7. 9; with slightly decreased intensities at lower pHs However, the pH 7.0 and lower phosphate buffers had no distinct minima visible at characteristic secondary structure wavelength values ( Figure 4 1a ). Comparison to background spectra l lines suggests that the peptide is partially structured T he CD spectra of KL 4 in the 4:1 POPC:POPG system (Figure 4 1b) showed uniform alpha helical peak characteristics for pH values of 7. 0 to 7. 9 ; though the intensity was lower in pH 7.9. At pH 6.7, c hanges in the minima peaks were observed ; t he lowest point on the left minima appears to have red shifted by 1 to 2 nm (209/210 nm); and the intensity decreased. At pH 6.4, a complete change in peptide conformation from alpha helical to

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100 beta sheet is obser ved. Though the minima value is at approximately 220 nm, not the standard 218 nm. After these experiments, a more accurate representation of the pH ranges (pH 6.4 to 7.9) was achieved with a Bis Tris propane buffer solution. The molar ellipticity values recorded of the peptide in the 4:1 DPPC:POPG lipid system were similar to that seen in phosphate buffer; though deviation from the alpha helical structure was visible at pH 7.3 versus pH 7.0 (Figure 4 2a). In the 4:1 POPC:POPG system, a gradual conformati on al change from alpha helical to beta sheet is recorded as the pH becomes more acidic. Conclusions We believe several factors contributed to the induced structural changes. Based on NMR and MD simulations of KL 4 within the selected liposome mixtures, i t has been concluded that the peptide selectively penetrates deeper into DPPC rich bilayers with distant electrostatic interactions between the charged lysine residues and the phospholipid headgroup s. 114 It is also know that the helical turns within this system are atypical in comparison to stan dard alpha helices ( i to i+4 ) Therefore, in decreasing the pH, it can be expected that a net potential gradient is created across the bilayer due to t he membrane surface potential being reduced. 115 For all experiments, the molar ellipticity ( ) values were considered qualitative observations. However, distinguishable shifts in relative minima intensities and wavelength were used as parameters to evaluate conformational changes. Due to elevated temperatures (45 C), the phosphate buffer acid dissociation constant increases creating a more acidic environment. Under such conditions, the overall surface charge density of both lipid systems would be reduced because

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101 ionisable phospholipid species such as POPG, have a higher probability of being neu tralized by solvent counterions. This directly affects the electrostatic interactions between the lysine groups of the peptide to the bilayer headgroup region; also affecting the net potential across the bilayer. With a lowered surface potential and the ch arges on the peptide having a greater affect within local area of integration bilayer expansion and reduction in curvature strains due to morphological strains are highly probable. In Figure 4 3a, the DPPC rich system shows a clear transition in peptide conformation between pH values 7. 0 and 7.3 Due to the abundance of saturated acyl chains, the natural packing properties of this system allowed for hydrophobic interactions with the peptide to retain structure. However, with lowering pH and bilayer expan sion, the structure of the peptide is no longer characterizable via CD measurements. In the fully monounsaturated liposomes (POPC:POPG) the general alpha helical structure is conserved over most of the pH range (pH 7.9 to 6.7). This is attributed to the d ifferences in fluidity of the hydrophobic acyl chains reducing the net effects of expa nsion on the peptide. However, a t pH 6.4 (approximately pH 5.8), a change from alpha helical to beta sheet is observed. Within the Bis Tris buffered DPPC:POPG liposomes the peptide showed similar conformational changes to that o f the phosphate buffering media; a general transition can be estimated at ~ pH 7.45 (Figure 4 3b). However in the more fluid POPC:POPG liposomes, conformational changes were visible at lower pHs (6 .7 and 6.4). The pH values at which dynamic changes in KL 4 secondary structure were recorded, overlap with known surfactant membrane interfacial pH values; between 6.5 and 6.7. These results highlight the importance of ionic interactions, surface potenti als

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102 and gradient potentials across the membrane on the surfactant peptide structure within its native environment. Though previous work has verified the alpha helical structure of the peptide in liposomes, the conformational changes seen at lower pHs could be an integral part of the mechanism of peptide function in the reduction of surface tension and/or lipid trafficking. Future investigations utili zing lower pH buffering systems will be necessary to truly model the dynamics of the alveoli subphase and may yield more accurate information on the dynamics associated with LS function. 116

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103 Figure 4 1. CD spectra of KL 4 in A) 4:1 DPPC:POPG and B) 4:1 POPC:POPG liposomes at 45 C Total lipid concentrations were 2 mM with 2 mol% peptide in a 10 mM NaH 2 PO 4 H 2 O /Na 2 HPO 4 buffe r solution with140 mM sodium chloride. Six buffer solutions of were made from one stock; pH adjustment to 6.4, 6.7, 7.0, 7.3, 7.6, and 7.9 were done with drops of 1 M sodium hydroxide. The step scan method was used. Data were collected every 1 nm with a fo ur second averaging and wait time in between scans. Each spectral line is an average of 8 scans.

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104 Figure 4 2. CD spectra of KL 4 in A) 4:1 DPPC:POPG and B) 4:1 POPC:POPG liposomes at 45 C Total lipid concentrations were 2 mM with 2 mol% peptide in a 1 0 mM Bis Tris propane buffer solution with140 mM sodium chloride. Six buffer solutions of were made from one stock; pH adjustment to 6.4, 6.7, 7.0, 7.3, 7.6, and 7.9 were done with drops of 1 M hydrochloric acid. The step scan method was used. Data were co llected every 1 nm with a four second averaging and wait time in between scans. Each spectral line is an average of 8 scans.

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105 Figure 4 3 Molar ellipticity ( ) at 222 nm as a function of pH values in 10 mM A) phosphate buffer and B) Bis Tris buffer solutions with 140 mM sodium chloride at pH 6.4, 6.7, 7.0, 7.3, 7.6 and 7.9. Each data point represents the

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106 CHAPTER 5 PEPTIDE EFFECTS ON MEMBRANE ORGANIZATION AND DYNAMICS Abstract KL 4 a 21 residue mimetic of surfactant protein B, is effective in the treatment of infant respiratory distress syndrome (RDS) and functions by lowering a l veolar surface tension and promoting oxygen exchange. H ere we utilized a pyrene phospholipid analog to investigate the effect of KL 4 on lipid organization and acyl chain dynamics by monitoring changes in excimer to monomer (I e /I m ) ratios ; and observed the ability of the peptide to modify membrane fluidity prop erties via anisotropy measurements of a rhodamine labeled phospholipid The pyrene phospholipid probes the local environment of the hydrophobic core of DPPC:POPG and POPC: POPG liposomes. As the concentration of KL 4 was increased (0.5 to 5 mol%), an average decrease of ~27 40% and ~0 10% in I e /I m was observed in the DPPC : POPG and POPC : POPG LUVs, respectively, which is directly proportional to a lowered probability of excimer formation, which is highly dependent on proximal interactions of an excited monomer with a pyrene moiety at ground state Changes in anisotropy and fluorescence emissions were observed with the rhodamine labeled phospholipid probes A steady increase in the order was observed in the DPPC/POPG liposomes with relatively constant fluorescenc e intensity, while collisional quenching was observed in the POPC/POPG liposomes. These observations agree with NMR observations and proposed mechanisms of peptide mediated lipid trafficking. Abbreviations used: POPC, 1 palmitoyl 2 oleoyl sn glycero 3 ph osphocholine; DPPC, 1,2 dipalmitoyl sn glycero 3 phosphocholine; POPG, 1 palmitoyl 2 oleoyl sn glycero 3

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107 phosphoglycerol; PPDPC, 1 palmitoyl 2 (pyrene 1 yl)decanoyl sn glycero 3 phosphocholine ; rhodamine PE, 1,2 dipalmitoyl sn glycero 3 phosphoethanolamine N (lissamine rhodamine B sulfonyl) ; DPH, diphenylhexatriene; TFA, trifluoro acetic acid; HEPES, 4 (2 hydroxyethyl) 1 piperazineethanesulfonic acid C 8 H 18 N 2 O 4 S; EDTA, ethylenediamine tetraacetic acid, C 10 H 16 N 2 O 8 ; NaCl, sodium chloride; NaN 3 sodium azide; NaOH, sodium hydroxide; PC, polycarbonate; MLV, multilamellar vesicle; LUV, large unilamellar vesicle; SP B, surfactant protein B; ssNMR, solid state nuclear magnetic resonance; RDS, respiratory distress syndrome. Introduction Lung surfactant ( L S) is a c omplex mixture of phospholipids and associated surfactant proteins produced by type II alveolar cells that coat the alveoli 19 and form a lipid rich barrier at the air fluid interface 55 117 L S is known to lower alveolar surface tension, enabling expansion of the lung during the inspiration of air and LS prevent s lung collapse during expiration 20 11 8 A deficiency in the production of L S leads to respiratory distress syndrome (RDS), a breathing disorder that remains a leading cause of death amongst preterm births. Therefore, research efforts are focused on the development of surfactant replacement th erapies. To date, bovine derived surfactants are the most widely used form of treatment for various chronic lung diseases (CLDs) 8 33 However, growing concerns about the potential risk of immunogenic and infectious complications 34 expense, supply limitations, and inconsistencies in surfactant composition due to extraction methods, have inspired research efforts towards the developm ent of purely synthetic options 35 36 37 38 39 Previous studies determined that the major phospholipid component (DPPC) 8 alone was not able to maintain PS functionality, demonstrating the necessity of

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108 lung surfactant proteins (SP A, SP B, SP C and SP D) 118 119 43 to retain activity. The hydrophilic proteins, SP A and SP D, are involved in immune defense, whereas SP B and SP C are critical for lipid organization and surfactant durability, enabling surface tension reduction 33 120 Gene knock out studies characterize SP component because lethal respiratory failure occurs in its absence. Expression of SP B and SP C in their native form poses challenge s due to their hydrophobicity and lack of stability, particularly with SP C 44 leading to the production of a number of synthetic analogs such as the following : mini B, SP B, KL 4 SP C33, etc 43 KL 4 a major component of Surfaxin 46 48 49 50 51 is the first synthetic peptide containing surfactant to be used in neonatal medicine, was designed to mimic the C ter minus of SP B. Alt h ough its amino acid sequence differs substantially from the protein fragment (Figure 1 9) similarity in its charge density and hydrophilic/hydrophobic ratio 55 are necessary for functionality. Several low resolution spectroscopic techniques such as Fourier t ransform i nfrared ( FT IR) and c ircular d ichroism (CD) spectroscopy 55 121 122 have been used to investigate the structure of the peptide in lipid environments Results from these investigations suggest KL 4 is helical, though there were conflicting reports concerning its orientati on relative to the bilayer normal (perpendicular/transmembrane) or in plane (parallel) to the bilayer surface More recently, solid state n uclear m agnetic r esonance (ssNMR) and e lectron p aramagnetic r esonance (EPR) studies, coupled with d ifferential s canni ng c alorimetry (DSC) and CD, have provided information regarding the penetration depth of the peptide within DPPC:POPG and POPC:POPG bilayers and have shown the orientation to be in the plane rather than transmembrane 55 The Long and Fanucci labs h ave showed that both

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109 partitioning and helical rotation angle of KL 4 depend on the level of fatty acid monounsaturation and curvature strain ; thus providing a mechanism to explain how DPPC is trafficked in PS. 114 Other methods such as s canning f orce m icroscopy (SFM), film balance, t ime of f light s econdary i on m ass s pectrometry (TOF SIMS) and fluorescence light microscopy, were used by others to monitor the phase behavior and other physical characteristics of monolayers upon peptide binding ; results showed that the presence of an anionic lipid was crucial for KL 4 integration into liposomes. 51 Despite the advances in elucidating structural properties of the peptide, molecular level information is still pertinent to understanding how it moderates surface tension in the alveoli To study localized environmental changes and lipid dynamics we utilized fluo rescence emission and anisotropy experiments exploiting pyrene and rhodamine phospholipid analogs 56 57 58 59 to track specific membrane protein interactions in relation to surfactant function. Both techniques present advantages over other high resolution techniques given the experimental time scale and low sample consumption ; then, making it a cost effective option. Material and Methods Synthesis of KL 4 KL 4 was prepared through aut omated solid phase synthesis on a Wang resin (ABI 430, ICBR, University of Florida). It was then cleaved from the resin with 90% TFA/5% triisopropyl silane/5% water and ether precipitated. The crude product was purified by reverse phase high performance li quid chromatography using acetonitrile/water gradient, and purity was verified by mass spectrometry. A KL 4 stock

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110 (Molecular Structure Facility, University of California, Davis, CA), was used for all experiments described. Preparation of L iposome S amples POPC, DPPC, POPC we re purchased as chloroform solutions (Avanti Polar lipids, Alabaster, AL) and concentrations were verified by phosphate analysis (Bioassay Systems, Hayward, CA). Lipid stocks were mixed in chloroform to obtain molar ratios of 4:1 POPC:POPG and 4:1 DPPC:POP G, and 3 mol% PPDPC or 0.05 mol% r hod amine PE depending on the type of fluorescent experiment being performed Aliquots of KL 4 0.5 to 5 mol%, were mixed with lipid stock solutions. Test tubes containing the peptide and lipid mixtures were placed in a 45 50 C water bath with a stream of nitrogen blowing over them to remove the chloroform/methanol solvent phase. The lipid peptide films were then reconstituted in cyclohexane, flash frozen, and lyophilized overnight to remove solvent. The res ulting lipid films were then hydrated at 45 C for approximately 1 2 hours (vortex mixing every 30 minutes) in 1 m L of a 5 mM HEPES, 0.1 mM EDTA, 100 mM NaCl, 0.02% NaN 3 (w/v) buffer solution (pH 7.4) to yield a total lipid thaw cycles and were left overnight in an incubator oven set at 45 C. LUVs were formed by extruding the MLV dispersions 31 times through a 100 nm polycarbonate membrane (Millipore, Bedford, MA) ; extrusions were performed on a heating block with the hot plate set at ~55 C Measurement of I e /I m for Samples Containing PPDPC Immediately after extrusion, samples of 4:1 POPC:POPG and 4:1 DPPC:POPG containing PPDPC and 0 to 5 mol % KL 4 were allow ed to equilibrate in a temperature controlled sample cell of the FluoroMax 3 fluorometer (Jobin Yvon Horiba, Edison, NJ)

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111 for 5 minutes at 4 5 C. The e xcitation wavelength was set at 344 nm, a known value for pyrene, w hereas emission data were collected f or wavelengths 360 550 nm. For all reported data, emission intensities are recorded for wavelengths of 395 nm and 480 nm, which are the pyrene monomer ( I m ) and excimer ( I e ) respectively Data were collected in triplicate, averaging 3 scans per sample; rep roduced 3 times with separate samples prepared from the same stock solutions Scans of the buffer were used for background subtractions. The excitation and emission slits were set to 5 nm, and the excitation and emission polarizers were set to 90 and 0 r espectively. Measurements were made using a 4 mm light path quartz cuvette (Starna, Atascadero, CA). Fluorescence A nisotropy of Samples c ontaining Rhodamine PE Similar to the methodology described for the measurement of I e /I m for PPDPC section, samples o f 4:1 POPC:POPG and 4:1 DPPC:POPG containing r hod amine PE and 0 to 5 mol% KL 4 were equilibrated at 45 C in the temperature controlled sample cell of the Fluoromax spectrofluorometer Polarized emission was measured in the L format setting the excitation and emission values for r hod amine PE at 557 nm and 587 nm respectively. Alt hough full scans of individual polarizer emission intensities were recorded for some samples, only single point anisotropy ( r ) measurements were reported at the aforementioned emiss ion wavelength. Details of instrumental set up are explained in Chapter 2. Results and Discussion Selective KL 4 P artitioning into DPPC:POPG bilayers Initi al studies with PPDPC in model liposomes (reported in Chapter 3) established that changes in the ex cimer to monomer intensities could be optimally detected at 3 mol % PPDPC in both lipid systems (DPPC:POPG and POPC:POPG) All

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112 spectra were collected at 45 C to ensure all samples were in the fluid phase b ecause of the high gel to fluid phase transition t emperature (T m = 35 C ) of the DPPC rich liposomes mixed with fluorescent probes, and to enable direct comparison of observations with previous work. F igure 5 1 shows a decrease in excimer formation is observed upon addition of KL 4 to both DPPC:POPG and PO PC: POPG vesicles; although the change is more prominent in the former. With the pyrene being attached to the tenth carbon of the phospholipid acyl chain in PPDPC, the decrease in I e /I m implies that insertion of KL 4 attenuate d interactions between pyrene mo ieties in the hydrophobic lipid core, suggesting the peptide resides in the vicinity (Figure 5 2) Changes in lipid movement and order due to peptide penetration into the hydrophobic core would directly impact the decay time of the monomer along with affec ting t he level of excimer formation. The more pre valent changes observed in DPPC: POPG vesicles correlate well with previous ssNMR and EPR studies suggesting that the deeper penetration of the peptide into DPPC rich systems leads to more ordering of the hyd rophobic core than is observed in m onounsaturated lipid mixtures. This result also highlights the sensitivity of th e pyrene assay to changes in the packing and dynamics of the hydrophobic core of lipid systems suggesting it as a simple, cost effective mean s for screening the interactions of SP B mimetics with lipid bilayers. Distinct differences in penetration depth have been correlated to hypothesized mechanism of lipid flipping to the air fluid interface for oxygenation due to induced curvature strains. Collisional Q uenching of H eadgroup T ethered P robe To probe the effect of KL 4 on lipid dynamics in the headgroup region, fluorescence anisotropy and emission of rhodamine ta gged phosphatidylethanolamine (r hod amine PE) was monitored as a function of KL 4 ad dition to DP PC:POPG and

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113 POPC: POPG liposomes. Based on preliminary control experiments a 0.05 mol% fluorophore concentration relative to the total lipid concentration, was chosen to minimize liposome disorganization on addition of the bulky chromophore A nisotropy values typically reflect the change in membrane free volume or the rigidity of the local environment; however, the ability of the rhodamine chromophore to fold back onto the polar headgroups contributes to the measured values. As shown in Figur e 5 3 an increase in the relative fluorescence anisotropy (r r 0 ) was observed upon i ntegration of KL 4 into both liposomes; where r 0 represents the anisotropy value at 0 mol% peptide, and r is anisotropy at varying concentrations of peptide. However, a la rge decrease in fluorescence emission of the rhodamine PE was observed in the POPC: POPG liposomes with peptide integration (Figure 5 4 ) ; a slight decrease was observed in DPPC: POPG liposomes (data not shown here). As Previous ssNMR and EPR work has found KL 4 associates more peripherally with POPC:POPG liposomes, which we believe orders the lipid headgroups, leading to collisional quenching and the observed decrease in fluorescence intensity. The polar nature of the chromophore allows it to flip back onto polar headgroup s of the bilayer, decreasing proximal distances. While KL 4 appears to facilitate retention of overall natural bilayer undulations would increase the chances of collisional quen ching. This effect is not seen in the DPPC:POPG system, suggesting the penetration depth of peptide is sufficient to cause a spread in the lower tail regions that could offset distance s between headgroups

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114 SP B is crucial in lung surfactant homeostasis, specifically functional trafficking of DPPC to the air water interface reducing surface tension. Studying the effects of clinical peptide mimetic of SP B, KL 4 on model lipid systems facilitates our understanding of the m olecular biophysics underlying L S. Here we have shown how relatively straightforward fluorescence assays using commercially available fluorophores and small sample sizes can be used to rapidly assay the effects of a L S peptide on lipid organization. These assays allow clear distinction be tween peptide effects on DPPC rich liposomes and POPC rich liposomes, suggesting they could be used in high throughput screening of other peptide mimetics of SP B. In future work, we will explore the effects of native SP B on model lipid systems using the se assays to examine whether similar changes in lipid organization are seen on addition of the protein. Conclusions SP B is crucial in lung surfactant homeostasis, specifically functional trafficking of DPPC to the air water interface reducing surface tension. Studying the effects of clinical peptide mimetic of SP B, KL 4 on model lipid systems facilitates our understanding of the m olecular biophysics underlying L S. Here we have shown how relatively straightforward fluorescence assays using commerciall y available fluorophores and small sample sizes can be use d to rapidly assay the effects of a L S peptide on lipid organization. These assays allow clear distinction between peptide effects on DPPC rich liposomes and POPC rich liposomes, suggesting they co uld be used in high throughput screening of other peptide mimetics of SP B. In future work, we will explore the effects of native SP B on model lipid systems using these assays to examine whether similar changes in lipid organization are seen on addition of the protein.

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115 Figure 5 1 Effects of KL 4 concentration on rate of excimer formation as shown with the percent change in excimer to monomer ratios (I e /I m ) in 4:1 DPPC:POPG ( hashed bars) and 4:1 POPC:POPG (solid bars) containing 3 mol% PPDPC. The perc ent changes were computed relative to initial I e /I m values at 0 mol% KL 4 (pH 7.4). Data were collected in triplicate at 45 C, with an excitation wavelength of 344 nm. Error bars represent the standard deviation of measurements from three different samples pr epared from the same stock lipid mixture. *Percent change was computed by the following: ((( I e /I m ) i ( I e /I m ) f ) ( I e /I m ) i ) 100 where i and f are the initial and final ratios, respectively.

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116 Figure 5 2. Hypothetical placement of PPDPC chemical struct ures on models 117 for the interaction of KL 4 with A) 3:1 POPC:POPG an d B) 4:1 DPPC:POPG liposomes Lipid coordinates were provided by Scott Fellers. 123

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117 Figure 5 3 Effects of KL 4 concentration on relative changes in anisotropy (r) of 4:1 DPPC:POPG ( hashed bars) and 4:1 POPC:POPG (solid bars) liposomes containing 0.05 mol% rhodamine PE The changes were computed relative to initial anisotropy (r 0 ) values at 0 mol% KL 4 Total lipid concentrations were 25 triplicate at 45 C, with an excitation wavelength of 557 nm and emission wavelength of 587 nm Error bars represent the standard deviation of measuremen ts from three different samples prepared from the same stock lipid mixture. *The anisotropy change was computed by the following: r r 0 where r is the value at n mol% (n = 1 to 5) KL 4 and r 0 is the value 0 mol% KL 4

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118 Figure 5 4 Effects of KL 4 concen tration on anisotropy (black squares) and fluorescence emissions (blue triangles) in 4:1 POPC:POPG liposomes containing 0.05 mol% rhodamine PE. HEPES buffer solution (pH 7.4). Data were collected in triplicate at 45 C, with an excitation wavelength of 557 nm and emission wavelength of 587 nm. Error bars represent the standard deviation of measuremen ts from three different samples prepared from the same stock lipid mixture.

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119 CHAPTER 6 ACCESSIBILITY STUDIES WITH SPB C AND SPIN LABEL ED LIPIDS Introduction Power saturation is a continuous wave electron paramagnetic resonance (CW EPR) technique that can be used to determine orientation and pa rt it ioning depth profiles of molecules in lipid bilayers, specifically by studying the solvent accessibility to a spin label within its local environment. Preliminary studies utilized 1 mol% spin labeled lipid probes (tempo POPC and n doxyl PSPC) within 4:1 DPPC:POPG liposomes to optimize expe rimental conditions, from which a 20 mM nickel (II) acetylacetonate (NiAA) at pH 6.5 and other instrument parameters were determined. Following these experiments, surfactant pepti de SP B 59 80 was incorporated into 4:1 DPPC:POPG and 4:1 POPC:POPG lipid systems containing spin labeled lipid probes, at varying concentrations (0 to 3 mol%) of the peptide. In comparison to previous work done with KL 4 peptide, the small changes in comput ed solvent accessibility ( P 1/2 ) and depth ( ) parameters for SP B 59 80 suggests its penetration is more peripheral in the liposomes and will serve as calibration profiles for future work with the spin label ed peptide. Material and Methods Synthesis of SP B 59 80 SP B 59 80 (DTLLGRMLPQLVCRLVLRCMD) was prepared through automated solid phase synthesis on a Wang resin (ABI 430, ICBR, University of Florida). It was then cleaved from the resin with and ether precipitated. The crude product was pu rified by reverse phase high performance liquid chromatography using acetonitrile/water gradient, which contained a 0.3% TFA; and the purity was verified by mass spectrometry. A double mutant of SP B 59 80 was expressed using a pET31

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120 construct (EMD Bioscien ces, Inc., Gibbstown, NJ) that incorporated a synthetic gene for the peptide (DNA2.0, Menlo Park, CA) in BL21(DE3) cells. This was followed by standard purification and cleavage protocols. Both methionines and cysteines were replaced by isoleucines and ser ines, respectively. A stock solution with a concentration of 0.818 mM 0.040 and later diluted to 252 amino acid analysis (Molecular Structure Facility, University of California, Davis, CA) was used to more accurately determine the final concentrations Buffer Solutions Protocol Two 5 0 mM Bis Tris propane (Sigma Aldrich, St. Louis, MO) b uffer solutions containing 140 mM sodium chloride were made at pH 6.5 and 7.5 10, 20 and 30 mM nickel (II) acetylacetonate hydrate ( NiAA ) solutions (Figure 6 1a) were prepared with the stock Bis Tris buffers measured with UV (Figure 6 1b and 6 2) and re frigerated. All stock buffer solutions with and without nickel were used to hydrate pre aliquoted dried lipid peptide films at 45 C with vortex mixing. Preparation of Liposome S amples for Nickel Determination POPC, DPPC, POPC tempo POPC and n doxyl PSP C were purchased as chloroform solutions (Avanti Polar lipids, Alabaster, AL) and concentrations were verified by phosphate analysis (Bioassay Systems, Hayward, CA). Lipid stocks were mixed in chloroform to obtain molar ratios of 4:1 POPC:POPG and 4:1 DPPC :POPG, and 1 mol% tempo POPC or n doxyl PSPC. For the nickel determination experiments, the lipid mixtures were hydrated with 0, 10, 20 and 30 mM NiAA buffer solutions at pH value s 6.5 and 7.5 with a 20 mM total lipid concentration T he suspensions underw ent 5 freeze thaw cycles and the M LV dispersions were placed in a gas permeable TP X capillary (Molecular Specialties, Inc. Milwaukee, WI) Each sample suspended in 5 0 mM

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1 21 Bis Tris buffer (0 mM NiAA) was purged and equilibrated in nitrogen gas or air (~20% o xygen) at 45 C for ~20 minutes ; samples hydrated with NiAA buffers were equilibrated under nitrogen gas. Preparation of Liposomes containing Surfactant Peptide Lipid stock solutions containing spin labeled lipid probes were made as previously described Aliquots of SP B 59 80 0 to 3 mol% (+1 mol% increments), were mixed with lipid stock solutions. Vials containing the peptide and lipid mixtures were placed in a 45 50 C water bath with a stream of nitrogen blowing over them to remove the chloroform/me thanol solvent phase. The lipid peptide films were then reconstituted in cyclohexane, flash frozen, and lyophilized overnight to remove solvent. The res ulting lipid films were then hydrated in 50 L of either 0 or 20 mM NiAA buffer solutions at pH 6.5 to yield a total lipid concentration of 20 mM T he suspensions underwent 5 freeze thaw cycles and the MLV dispersions were placed in a gas permeable TPX capillary (Molecular Specialties, Inc. Milwauk ee, WI) Each sample suspended in 5 0 mM Bis Tris buffer (0 mM NiAA) was purged and equilibrated in nitrogen gas or house air (~20% oxygen) at 45 C for ~ 20 minutes; samples hydrated with NiAA buffers were equilibrated under nitrogen gas Temperature regula tion was implemented by passing gasses through a copper coil in a recirculating water bath set at 61 C. CW EPR Spectra Acquisition Spectra for all samples were collected on the Bruker ER 200 spectrometer (Billerica, MA), which has been modified with a lo op gap resonator (Medical Advances, Milwaukee, WI) using a 2 mW power level During the period used for sample equilibration, the spectrometer was tuned and parameters were set Initial spectra were

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122 collected with a sweep width of 100 Gauss and the modulat ion amplitudes were set based o n the spin labeled lipid probe; for samples containing tempo POPC and 12 doxyl PSPC probes the modul ation amplitude was set to 7 Gauss; for the 5 doxyl and 7 doxyl PSPC probes, the modulation amplitude was set to 10 Gauss A s tandard parameters listing is shown in Table 6 1. From the initial scan a center field set based on the central resonance line and all subsequent scans were done with a 20 Gauss sweep width. Power Saturation Experiments Spectra were collected over th e power range of 0.2 5 to 39.9 mW. The peak to peak amplitude ( A pp ) for the central line were measured (Figure 6 3) and then plotted against the incident power to extract the P 1/2 value, the power at which the intensity of the central line is hal f of its un saturated intensity. T he amplitudes are fit to the following expression (Equation 6 1) in equation to obtain the P 1/2 value, where I is the scaling (6 1) factor, P is the microwave power, and is a measure of the homogeneity of the resonance saturation ; data fitting was done using LabView software (National Instruments, Austin, TX) provided by Christian Altenbach and Wayne Hubbell (UCLA, Los Angeles, CA) P 1/2 values are recorded under three conditi ons: (1) where the samples without nickel are equilibrated in diamagnetic nitrogen gas as the background (2) those same samples are equilibrated in air (20% paramagnetic oxygen), and (3) the samples hydrated with the NiAA buffer are equilibrated in nitrog en. The non polar oxygen diffuses i nto hydrophobic bilayer and the water soluble paramagnetic nickel

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123 collider concentrates in the polar headgroup region. The P 1/2 values are used to calculate depth parameters ( ) as shown in the following equations: (6 2) (6 3) Where the collider values are from either paramagnetic species (oxygen or NiAA). Additional collision parameters can be computed but will not be shown here. Results and Discussion Nickel Determination Experiments Power saturat ion experiments require the use of paramagnetic colliders such as a water soluble metal complex or non polar molecular oxygen, to interact with a spin label via the Heisenberg exchange described in Chapter 2. The enhanced relaxation effects of the electron magnetic dipole moment transitions provide information about the accessibility of the collider to the spin label. House air containing ~20% oxyg en, is the paramagnetic collider that diffuses in to the hydrophobic regions of the lipid bilayer; the water soluble metal complex concentrates in the polar heagroup regions As lipid bilayers have a potential gradient, it is expected that a concentration g radient of the paramagnetic colliders will be formed; therefore the relaxation effects measured will vary with the location of the spin label To choose sets of conditions where these differences in relaxation effects would be enhanced, the following NiA A concentration were chosen: 10, 20 and 30 mM, at pH values of 6.5 and 7.5. As described previously, the P 1/2 values were used to calculate depth parameters as a function of the spin label location on the lipid probe; the data for pH 6.5 are shown in Figur e 6 4. For liposomes containing the tempo PSPC in 30 mM

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124 NiAA, an arbitrary saturation value of 100 was used to calculate the accessibility parameters because a P 1/2 value could not be reached within the incident power range used. The preferential localizat ion of NiAA at the headgroup region would cause an increase in collisions with the tempo spin label, leading to higher relaxation rates; all other values were based on experimentally extracted P 1/2 values. The 7 doxyl PSPC measurements did not follow the t rends of other spin label positions. Based on the data collected, using the 20 mM NiAA buffer at pH 6.5 was chosen for experiments that followed. Accessibility Measurements with SP B 59 80 Assimilation The solvent accessibility parameter ( 1/2 ) for NiAA was plotted as a function of mol% SP B 59 80 in the 4:1 DPPC:POPG and 4:1 POPG liposomes and with varying spin labeled positions (Figure 6 5). Prior to addition of the peptide, NiAA is shown to be equally accessibility to the tempo, 5 and 7 doxyl positions in the DPPC rich liposomes; and shows changes between the tempo, 7 and 12 doxyl positions in the POPC:POPG liposomes. However with incorporation of the peptide (1 to 3 mol%), the 1/2 values began to overlap at all positions with the ex ception of 12 doxyl. Figure s 6 6 and 6 7 show plots of the 1/2 values for oxygen and NiAA according to th e spin label position; all values appear to be within error in both lipid systems. To monitor the general effect of SP B 59 80 on spin label depth, the depth parameter ( ) was computed and plotted as a function of increasing peptide concentration. The values are negative for the 5 and 7 doxyl positions which is an indication that there is higher accessibility of the NiAA collider than that of oxyge n. For the DPPC rich lipid, there is a slight increase in values at the 5 doxyl position with increasing peptide concentration, which suggests the spin label may have moved

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125 slightly deeper into the hydrophobic core. H owever for both lipid sy stems, there was no net change observed in the 7 doxyl position. The 12 doxyl position data shows a shift to more positive values with peptide incorporation for both lipid systems. Conclusions From the nickel determination experiments, distinct changes in the value s were not observed at pH 7.5; and with difficulties in obtaining P 1/2 values at the tempo position for 30 mM NiAA, a final condition of 20 mM NiAA (pH 6.5) was determined to be the optimal condition for the power saturation CW EPR experiments. Despite ins ignificant changes in the accessibility and depth parameters, the data suggest the peptide does not partition deeply into the liposomes contrary to previous KL 4 data. The current work will be used as calibration profiles for future work involving spin labe led SP B 59 80

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126 Figure 6 1. A) Chemical structure of nickel (II) acetylacetonate (NiAA) used to make buffers for power saturation experiments. B) UV spectra of 0, 10, 20 and 30 mM NiAA buffer with 50 mM Bis Tris propane + 140 mM NaCl buffer at pH 6.5 ; inset shows peaks in the wavelength range of 500 to 850 nm. Figure 6 2. Standard curve of 10, 20 and 30 mM NiAA buffers made with 50 mM Bis Tris propane + 140 mM NaCl buffer at pH 6.5.

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127 Table 6 1 Standard CW EPR parameters used for power saturation experiments Parameter Value Number of points 1024 Number of scans 1 Center field 3255 3276 Gauss Sweep width 100 or 20 Gauss Acquisition time 41.943 sec Frequency 9.3 9.4 GHz Power 0.25 39.9 mW Receiver gain 10 4 10 5 Modulation amplitude 7 or 10 Gauss Modulation frequency 100 KHz Time constant 0.16384 sec Phase 100 deg 3 Figure 6 3. CW EPR spectrum of a standard nitroxide spin label showing three transition peaks. The peak to peak ( A pp ) amplitude is measured and used for power s aturation curve plots.

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128 Figure 6 4 Depth parameter plotted as a function of the followi ng spin label ed lipids: tempo PO PC, 5 7 and 12 d oxyl PSPC (1 mol%) 10, 20 and 30 mM NiAA buffers made with 50 mM Bis Tris propane + 140 mM NaCl buffer (pH 6.5 ) were used to hydrate the 4:1 DPPC:POPG liposomes and to determine the optimal nickel concentration for power saturation experiments. Total lipid concentration were 20 mM and a ll measurements were made at 45 C. Figure 6 5 Power saturation accessibili ty parameter P 1/2 (NiAA) plotted as a function of mol% SP B 59 80 in A) 4:1 DPPC:POPG and B) 4:1 POPC:POPG liposomes. Each contained 1 mol% of either tempo POPC, 5 7 or 12 doxyl PSPC probes and were hydrated in 0 or 20 mM NiAA buffers made with 50 mM Bis Tris propan e + 140 mM NaCl buffer at pH 6.5; total lipid concentration were 20 mM Data were collected in triplicate at 45 C; error bars represent the standard deviati on of three measurements from aliquots of the same sample

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129 Figure 6 6 Power saturation accessib 1/2 (O 2 ) plotted as a function of mol% SP B 59 80 in 4:1 DPPC:POPG and 4:1 POPC:POPG liposomes, which are separated according to the following spin label ed lipid probes: A) tempo POP C, B) 5 doxyl PSPC C) 7 doxyl PSPC or D) 12 doxyl PSPC ( 1 mol% ) Liposomes were hydrated in 0 or 20 mM NiAA buffers made with 50 mM Bis Tris propane + 140 mM NaCl buffer at pH 6.5 ; total lipid concentration were 20 mM Data were collected in triplicate at 45 C; error bars represent the standard deviation of th ree measurements from aliquots of the same sample.

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130 Figure 6 7 Power saturation accessibility parameter P 1/2 (NiAA) plotted as a function of mol% SP B 59 80 in 4:1 DPPC:POPG and 4:1 POPC:POPG liposomes, which are separated according to the following spin label ed lipid probes: A) tempo POP C, B) 5 doxyl PSPC, C) 7 doxyl PSPC, or D) 12 doxyl PSPC ( 1 mol% ) Lipo somes were hydrated in 0 or 20 mM NiAA buffers made with 50 mM Bis Tris propane + 140 mM NaCl buffer at pH 6.5 ; total lipid concentration were 20 mM Data were collected in triplicate at 45 C; error bars represent the standard deviation of three measureme nts from aliquots of the same sample.

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131 Figure 6 8 Depth parameter plotted as a function of mol% SP B 59 80 in 4:1 DPPC:POPG and 4:1 POPC:POPG liposomes, which are separated according to the following spin label ed lipid probes: 5 (left) 7 (center) or 12 doxyl (right) PSPC (1 mol%). Liposomes were hydrated in 0 or 20 mM NiAA buffers made with 50 mM Bis Tris propane + 140 mM NaCl buffer at pH 6.5 ; total lipid concentration were 20 mM Data were collected in triplicate at 45 C; error bars represent the standard deviation of three measurements from aliquots of the same sample

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132 CHAPTER 7 CONCLUSIONS AND FUTURE DIRECTIONS The effective restoration of lung compliance and surface tension reduction by Surfaxin, a therapeutic agent used to treat infants with RDS, makes it an ideal system to study fundamental protein lipid interactions and their roles in lung surfactant metabolism. The low molecular weight and stable KL 4 peptide a mimetic of SP B and component of Surfaxin, allows a direct cor relation of changes in membrane bilayer dynamics and fluidity to specific function s of SP B The scope of the work included in this dissertation evaluated the effects of KL 4 on liposomes composed of abundant LS phospholipids : DPPC, POPC and POPG. Two flu orescent techniques pyrene excimer to monomer emissions and anisotropy were utilized to the study the effects of LS peptides on membrane dynamics and fluidity. While integration of probes into biological membranes is common, we have shown it is crucial t o characterize their localization and orientation within bilayers as this directly affects the information extrapolated from recorded data (Chapter 3) CD measurements of the peptide structure within bilayer suspensions of varying pH proved the significan ce of mem brane bilayer surface potential and interfacial pH on pep tide conformational changes; factors that must be considered to further understand the complex mechanism of LS function The results from the pyrene emissions data were consistent with pri or work which indicate s peripheral association of the peptide in POPC:POPG liposomes equating to less dynamic changes in the pyrene emissions spectra. Through the I e /I m data, it is clear that KL 4 selectively penetrates deeper into DPPC rich liposomes res ulting in interference with the distance profiles of excited monomers to ground state monomers

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133 The collisional quen ching observed from rhodamine PE anisotropy measurements, yielded information on the collective dynamics of the liposomes relative to the me mbrane surface suggesting lipid re organization. The significance of p eptide electrostatic interactions with the polar head region, penetration depth, orientation and packing within lipid bilayer, have been observed. However to fully understand the func tional role of surfactant proteins and their effects on local bilayer dynamics in LS further experimentation is required. CD measurements on a modified KL 4 by reducing the net charge of the peptide by lysine replacement and experiments where the ionic s trength is varied, could be used to monitor the effect of pH and surface potentials on conformational changes. Deuterium NMR could be used to track chemical shifts the lysine residues within respective bilayers Though fluorescence techniques were utili zed to study the effects of KL 4 and SP B 59 80 on the lipid membranes, and currently SP N 1 25 applying these techniques to more complex syst ems that are more representative of the native LS composition could potentially aid our determination of each compo nents specific function. Previous NMR work has evaluated liposomes of 8:2:1 DPPC:POPG:Chol, 10:6:3:2:2 DPPC:POPC:POPG:POPE:Chol (synthetic CLSE), and a Surfaxin mimic composed of 3:1:2 DPPC:POPG:PA. By incorporating probes into these systems, we could moni tor induced phase separation events and correlate them with the previously described techniques. We would also like to use NBD PE, another fluorescent probe with rhodamine PE to monitor possible membrane fusion events triggered by lung surfactant peptides

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146 BIOGRAPHICAL SKETCH Otonye Braide received her B.A. in Chemistry, with a minor in Pure and Applied Mathematics, at L incoln University ( Lincoln University, PA) in 2004. She then obtained her M.S. in Chemistry from Georgia Institute of Technology (Atlanta, GA), with a research focus in simulating electrochemical processes via Cyclic Square Wave Voltammetry (CSWV) using MA TLAB. In the summer of 2007, she enrolled in U niversity of F lorida (BOE) program and then worked with Dr Charles R. Martin to develop a quantitative label free protein sensor using nanoporous alumina membranes. By 2010, she joined Dr Gail E. search group and in the summer of 2011, she began working to apply fluorescent techniques to the study of lung surfactant system dynamics and fluidity, a collaborative project with co advisor, Dr Joanna R. Long In the fall of 2012, s he began learning electron paramagnetic resonance (EPR) techniques for the determination of lung surfactant peptide orientation and penetration depth in lipid bilayers, not included in this dissertation. She received her Ph.D. from the University of Florid a in the spring of 2014.