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Stability and Functional Studies of Oligomeric M2 Ion Channels in Synthetic and Natural Lipid Environments



Permanent Link: http://ufdc.ufl.edu/UFE0007067/00001

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Title: Stability and Functional Studies of Oligomeric M2 Ion Channels in Synthetic and Natural Lipid Environments
Physical Description: Mixed Material
Copyright Date: 2008

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Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0007067:00001

Permanent Link: http://ufdc.ufl.edu/UFE0007067/00001

Material Information

Title: Stability and Functional Studies of Oligomeric M2 Ion Channels in Synthetic and Natural Lipid Environments
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0007067:00001


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STABILITY AND i TIONAL STUDIES OF OLIGOMERIC M2 ION CHAN i i IN
SYNTHETIC AND NATURAL LIPID ENVIR(i "MENTS











By

FRANK JOSEPH RAUCCI


A THESIS P. 7N'-TED TO THE GRADUATE SCHOOL
OF THE 141 *..ERSITY OF FLORIDA IN PARTIAL FULFILL I i T
OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


2004














AC; '\NOWLEDGEME

First, I would like to thank my committee members, .........:- Long, Arthur
r.1..;::: and Thomas DeMarse for their g,....1::. :... advice, and support. I would also

like to thank Randolf Duran and Peter Anderson for their advice .. :: the course of

this :: i: Their 1:: .1. was invaluable. 7 :. I would like to thank all the students

and post-doctoral members of the groups involved in this project for their help in data

:::and analysis. This work could not have been ,: :1: :'.1 without the help

of Kun r,. ... Jun Zhang, Frank :'.K Chris Williams, Brian Dorvel, Joy Wattawa,

and Becky Price. I would also like to thank my .: .. = ... for their love and support. I

would like to thank my friends for their -...... 1. 1 .. :..;I some ..-. i.:. i.. and

I would like to :" r; =: ly thank T. r': =: Cullem and Kaveri Marathe. And last but

certainly not least, I would like to thank my fiancee, Kalyani Marathe, for her love and

1. :.. I :: :::. ,,.:::: :: this very hectic time. I could not have, ::=i l,'",',i this work

without her.















'... -,=: .:: t of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of .. i.:.,:: of Science

STABILITY AND FUNCTIONAL STUDIES OF OLIGON.' 7" M2 ION CHAN' iS IN
SYNTHETIC AND NATURAL LIPID ENVIRCT I" TFNTS

By

Frank :.1. Raucci

December 2004

Chair: Thomas DeMarse
..D Biomedical 1


The self-assembly properties of the ion 1: ::.:: : i=. 1.. sC ':= .:.: of

the nicotinic acetylcholine :... '. i were examined at the air-water interface. The

... ', of monolayers formed by the i.i '- alone were c ... : .. to mixtures of

M2 with a synthetic lipid, 1,2-diphyanoyl-sn-, ---..'ro-3-phosphocholine (DPhPC) and

the1 1. I and i. i= : 1. alone viaT ::. = : ,=:-Blodgett surface pressure-area isotherms and

Angle *.i:: ::..., ...'. The DPhPC monolayer remains in a .;: : 1- phase until its

a. .w..*... pressure whereas the r monolayer undergoes two distinct :1: .. transitions

. .-.: -. collapse. M2 ion channel fluctuations were studies via tip-d:: .:1 patch clamp

methodologies with DPhPC and native 1 '..1 environments. Single channel fluctuations

were recorded and evaluated to determine the stochastic : .,.. .::.,. of the ion channel

to .'. .1 voltages. The results show that the M2 ion channel is ... .... .1 and

relatively stable. The pertinence of these results to 1:.:: !. ;:::. the .,.:- i. *:! y and

function of this ion channel in synthetic membranes .i il be discussed.













CHAPTER 1
INTRODUCTION

Project Overview

The immediate goal of this project is to study the effects of :. environment

on the -..... .. iles of a synthetic ion channel incorporated into a lipid : ..*. ...... This goal

:::::: passes achieving lipid '.. : ,....; membrane resistances greater than 106'Q cm2,

quantifying channel opening response times, and measuring single-channel current

fluctuations at low channel concentrations. The selected ion channel is the pore-

forming :.. 6-subunit of the nicotinic acetylcholine receptor, which is present in many

biologically relevant systems.

The ultimate goal of this .: .: is to use surface immobilization and

encapsulation within : ..c. mer-modified, tethered .i 2. ,: = ... in order to engineer

thin films of oriented, adds ...:- ., ion, i i The channels would then be used as

small biosensors that could detect minute amounts of a :.!:.. : :... agonist in aqueous

solution. .. : i .**. microelectronic detection methods for channel function might

include ion-sensitive microelectrode arrays (MEA's), field effect transistors (FET's), or

microelectrodes as extended gates of FET's.

The fundamental principles governing ion channel behavior in synthetic

membranes elucidated from our studies could be extended beyond the initial ion

channel being characterized in this work and may thus be..: A ..-. to a variety of

biosensors incorporating biological and synthetic ion channels with myriad functions,

physiological or otherwise. The possibility of improved medical diagnostics for a

number of toxicity-causing agents that affect the ion flow across i ii: ::: membranes in

humans may be realized by integrating the ion channels with electronics. Applications

are also ., : 1 in agricultural and military settings, as both would benefit from

1











a valuable diagnostic that could offer ::. ,:='.. detection of excessive acetylcholine

concentrations at the nicotinic-acetylcholine receptor caused by .....:. hosphate

*. ,..::: =. or other agonist interactions. Also, such a device could possible be extended

as a potent ,'. ....: ve to tissue harvest or human and animal research in areas such as

. 1:: ....... : :-:... The potential for ::: throughput assays of the i.. :::-: of new chemical

entities on ion channels also exists for functional biosensors.

As the nicotinic acetylcholine ::*: .',: :: is ::,* .. :::. in many tissue .. including

neurons and muscle, ... .. .. 1 ....: how the channel mechanism is by the

surrounding ~* .1 environment could also have a more direct medical :: : ::. Nicotine

and several anesthetics are numbered .......... the known ,.. .....: and ...- i of

nAChR which makes this :: ,*:: an attractive model for st ::: -. ':- transduction.

Ion Channels

Ion channels :A -..' an >..:. : :1 role in many 1- -:1. *.-:1 functions, ..:: 1::: .::: nerve

and muscle activity (Hucho and Wiese, 2001). Virtually all membranes in 1: .. cells

. .. .' a *...= : .: ::: of potential across them due to an ::: ::. : .:1 distribution of ions

in the intracellular and extracellular domains. This .. ... separation creates voltage

which is termed membrane potential. These potential differences -**: i'::, ::::. yet

distinct roles in different biolo'.-.-,1 cells. For instance, nerve cells use -- ....... .: .

.:1. called action i: ::::1 ;.:1'. that travel the length of the nerve axons to communicate

with other nerve cells at ......, tic .::... !: n ms. The value of the potential difference across

the .. membranes of any cell is :..- : by the Goldman-Hodgkin-Katz Equation,

which is a derivation of the Nernst equation. This equation .' .....- on the relative

permeabilities, Px, and the :: !, -::: : 1 [X]i, and external, [X]e, concentrations of the

respective ions present as well as the temperature and the gas constant, R, and the

Faraday i ..: :: =' F (Hille, 2001). Note that unlike the Nernst equation, the zero-current







3
potential in this case is in fact non-zero and represents a steady-state movement of ions

away from equilibrium.
RT i [ ..:]e + PK [K+]e + PcI [Cil]i
E = ---- In ------------------------------------------
F PNa [Na']i+ PK [K+]i + Pci [Cll]e


The1 i 1 :: 3 .: ; of plasm a m em branes .:: -. .ide a ...:.:= .. .:: ..::. .. barrier

to the movement of i.... ...-. particles from the intracellular to extracellular domains

and vice versa, thus : ::... ":::. for the establishment of these membrane .. ,.,.:I: :. In

order to 1.. .. ..1 ions between the two ionic reservoirs on either side of the plasma

membrane, the cells make use of -..' = : .. gated transmembrane proteins, known as

ion channels, to ... *i.*..- the ion currents.

Ion channels have many distinct features that make them of interest for

.:. and physiolo '. study. The first characteristic :... ... i. of ion channels

is their high ion selectivity. Channels not only show selectivity between anionic or

cationic molecules but also between monovalent and divalent ions or even for a specific

ion, such as H+(Hille, 2001). Secondly, ions channels are dynamic proteins, which

demonstrate identifiable responses under specified conditions. At resting potential,

the channels are completely closed and impermeable to ion current flow. However,

they can be opened, or gated, by either a change in the i:: .: potential or the

binding of i ..... = ligands (Hucho and Weise, 2001). Thus the two important

functional components characteristic of all ion channels are a selectivity filter that

determines which ions will be allowed to :: :- -" through the channel pore and a

gating mechanism that controls the opening of the pore. The two major classes of

ion channels are described by their gating mechanisms and thus all channels can be

classified as either voltage-gated or ligand-gated. Ligand-gated channels undergo a

conformational :: ..,. when a ; :. 1 : agonist binds to a receptor site. Voltage-gated

channels .: :::V .ii undergo a conformational change within the lipid I::. ::: i.:::- due to







4
a ;... .. .', usually polar si .. .....! of the structure..: ......, within the electric field

between the two lipid layers of the membrane bilayer (Hille, 2001).

,, -.otinic Acetylcholine Receptors

Channels are further classified into families and ".: ,-::: based on

functional and .-... i:. characteristics. There are three superfamilies of :. .*

gated channels: the ATP-gated purino receptors, the ion, !:. .::'. glutamate re(, :

and receptors that are similar to the nicotinic acetylcholine .. -- j.*., 2001). The

last ..: ::: which will be our primary focus, includes the nicotinic acetylcholine

receptor (nAChR), as well as glycine receptors, GABAA receptors, and some serotonin

receptors (Hucho and Weise, 2001). These c..::: :: .. *. have s:::: :: ::: sequences and

many conserved str :.: ::.: features. They all share s:::::: i.:' glycosylation patterns,
.-: L-. peptide loops created by disulfide bridges, similar distributions of prolines,

and conserved serine and threonine polar residues in the channel-forming domain

(Hucho and Weise, 2001). The most important feature shared by all of the receptors in

this superfamily is the presence of four i:: ...:.... i::..I amino acid sequences that span

the 1:1 : :::: .: i::, ::: :: ... labeled Ml through M4.

The pore of the nicotinic acetylcholine receptor (nAChR) was the first channel

pore to be characterized : : =: mutagenesis. These studies identified the residues of

the pore-lining segments and also i ::.: .,: :- :: ,d the selectivity filter function of the

channel was described. The functional form of nAChR is <::::: posed of five homologous

subunit proteins -, !::::.. : each i:::..- the four hyd :. i.::.i : transmembrane segments (Ml-

M4) that are characteristic of their superfamily. The nAChR is found in many cells,

including those responsible for signaling in neuronal and ::::: ::.:; tissues, and can be

composed of slightly different homologues. The stoichiometry of the nAChR varies

significantly depending on where the channel is found. In muscle tissue, nAChR has

a ... :: ..:.: of c2'3P'Y'6 while neuronal nAChR is a : ..:: i;'. ': ... ::"': : of a

subunits (Oblatt-Montal et al., 1993). As its name ::: :. ;* .. the nAChR is a 1 :.:::: -gated









the sequence of this s .. ....... is
different *.:'. The actual :: :. : :::: .: .i:. : :::: is small, only 23 to 25 amino acid

residues in 1. ... ...., and is modeled as an a-helical secondary structure based on electron

microsc(. studies of intact nAChR. This structure has recently been confirmed for

lone ... segments self-assembled in lipid ...: ..... i by two-dimensional solid-state N'..;'

(O=-11: etal., 1999). The '.' segments are also :::::: = 1:,: ::: in nature and they come

together at a ..-.. ,.... to form the funnel-like pore (Hucho and Weise, 2001).

The h- :'. 1:: : portions of the helices face the lipid :: 1 ::' and act to anchor the

... :.-. in the bilayer while the hyd ....... portion lines the pore region. When gated

open, there is a conformational, :: ::. .. in the orientation of the M2 segments, .

permeation of ions t= *..... the pore. The channel is selective for cations although it

does not discriminate between monvalent cations such as K' or Na' and even allows

permeation of Ca2+ to some extent (Hille, 2001). Studies have demonstrated that the

vestibules of the nAChR channel are :. '.' -: '1 : i:... :.. : .. the observation of

0.: ... -,. ..'.. .. ;'. Studies :: : -:"' nAChR samples from the electrical organ of T...'.

S.. .. and bovine calf muscle tissue have shown that the 6-s. '::: is the sole

determinator of the conductance \: .. .:=: :: of the nAChR in the :: ; = tissue ,

(Numa, 1989). Thus, when bovine a, [, and y subunits ;T: a T. .. 6-subunit

were incorporated into the bovine muscle tissue, there was a ::: : decrease in

conductance than when bovine 6 and T ... '. .... a, p, and y subunits were used.

. i::: ithe main portion of the i: I a-helices that form the ":' itself are ::.:..::. d, the

segments have, i: :: -.1 residues at either end which form -- rings of a sort when

the peptides are assembled into a pentamer. There are three : .:::- rings *:. -, : in the

full :. =..=:, one on the extracellular side of the "' T: sequence, one on the intracellular

side, and one intermediate within the actual sequence itself (Revah et .' 1991). It has

been shown by systemic mutation ...: ::. -: -= .- that channel conductance is diminished

as the net: 1:.:: : of one of these : ":::. .. becomes less ::: ..:!.:*ve (Imoto et al., 1988).







8
there were several ., ...:..:.. made in -.- ...... ... these structures. The solution

NMR structure differs from that of the solid-state structure in that it shows a kink in

the helix at leucine-11 (Breed et .:. 1996). This residue is thought to be important in the

: of the channel.

The.. -.:-1 ....:. nature of the individual *-- ;..1-: is I .1 A- to be the .

force behind the self-assembly of M2 helices into voltage-gating channels. Looking at

the sequence of the M2 6-subunit we ,'* ." .: see the: ........ of the individual amino

acid residues .'1: -.' an i:: =::: role in the function of the channel. The ..: ..

particularly the OH-i ..... ; serine and threonine residues, line the pore and play a

role in ion affinity and stabilization. The ::::::*. 1,::, b. .:1:::i;V: amino acid residues in

the sequence act to 1 .. -. the protein in place in the 1: 1 environment, which leads us to

......" :: : the effect of ::.1 selection on channel ]:

Lipids

S:.:.... are biological molecules that are insoluble in aqueous solutions and

soluble in .... solvents. Lipids serve not only as structural .. ......... of biological

membranes but they also :: :, reserves in the form of triacy; 1'..-:v'ols

and serve as vitamins and hormones in vivo. The membranes made up by 1 .:- are

assymetrical, fluid structures ..' 1 of fusion and are selectively permeable (: ::

et -.... 2002). They enable ....:. .>:nm of cellular or intracellular functions which by

variation in '. and protein .:::::: position, including the transmembrane proteins that

form ion channels. The .. of membranes are fatty acids, ..." .......

sphingolipids, and cholesterol. Phospholipids form the major ::1 ,'* part of biolo-. i:- 1

membranes and are composed of two fatty acids plus a .'.. : .'1 .. attached to a

glycerol. T ::.. I1: :: ::'. are like I-i '.ycerides .. .'Ithat the first hydroxyl of the

S.....molecule has a polar phosphate-u .. .. ... in place of the ;.:: acid.

This means that '1:: 1:::. i have ab f.1:. 7: head and 1: -.1::: '1::- i tail. This fact

forms the basis for the formation of self-assembled .... :.'. bilayers or micelles









in aqueous environments. The head group has a ...... that is specific for different

..1 1 ,. 1'... This head group will differ between cell membranes or different

concentrations of ... .. head groups. The .' y acid tails can also differ in :th and
,= :, but most usually have one saturated and one unsaturated leg of the tail.

. : ..... ....:.1 are a s'. of phospholipid derived from .. .... instead of glycerol.

Cholesterol is a :: ": :: :: : :: :::,.:. of cell :::: : :1.: :,., '. : .. ::1y those of eukaryotes,

and intercalcates between the fatty acid 1i ..'.. thus I ........them :..:>.. and

increased lateral fluidity (P= *:::.-= -zu et al., 2002). Cholesterol also makes the membrane

less permeable to small, water soluble molecules and it has been shown to thicken

1.... *" .... .= =. .:.= upon insertion.

With ..... to the nAChR, it has been shown that the optimal channel function

... .. .... the '=. : ... of negatively- .. .... .1 .,. 1. and cholesterol (Sunshine and

McNamee, 1992). For this project, *1: 1...'tanoyl-based lipids were chosen as they work

well for '.1 =::: ..: systems, such as the ones we are using, as well as the fact that they may

allow for more highly resistive *... .... formation than natural lipids due at least in part

to the :: ..:::: methyl groups on the fatty acid chains of the synthetic !:'. .'- They

are also well-suited for the -.. ..........- that will eventually be used to increase

stability in the electronic devices. The synthetic 1 !: I.. used in all =,.= =::==::: :::... was

the diphytanoyl derivative 1,2--. .1.A tanoy! -: .-. ycero-3--........ 1I.1:.. (DPhPC)

:,= :.. by Avanti Polar r .". whose structure is .:.::: to a native in F>:i .

3. As can be seen, DPhPC is ..... .i.. different from the native 1- associated wth

nAChR. Thus we undertook a thorough study of the channel f:: :::;... characteristics of 'V."

in DPhPC in order to establish the feasibility of I ... .. .. the lipid environment for i :.

sensor d. .L::: without ,:::":; :::::: ..::: the functionality of the ion channel.









a. 0

H 0


Electrical Properties of Membranes






H9 0

Figure 3: Comparison of Synthetic and Native Lipid Structure. Notice that the
fatty acid chains in POPE (a) are unmethylated whereas the synthetic DPhPC (b)
is methylated contributing to the additional rigidity of the bilayers they form.

Electrical Properties of Membranes

When attempting to measure the ease of current flow across a membrane,

whether due to pores in the membrane or functional ion channels, it is important to

understand the physical preasinciplg the governing the phenomenon. The ease current

flow is referred to as conductance and is usually measured between two conducting

electrodes in a salt solution. The conductance is governed by Ohm's Law, which states

I = gE

where I is the current, g is the conductance, and E is the potential difference across the

conducting material, in our case the ion channel. The conductance can be affected in

many ways by either changing the properties of the environment or the conducting

material. Conductance is increased by increasing the salt concentration using more

mobile salt ions, or decreasing the distance between electrodes. Conductance can be

decreased by introducing a nonconducting material between the electrodes, moving the

electrodes apart, or by increasing the viscosity of the ion salt solution. In the case of the

membrane ion channel system, the lipid bilayer acts as a barrier to ion flow and thus

decreases the conductivity or reciprocally increases resistivity of the system. Another









important consideration is the fact that a ? 1 bilayer membrane has a .'.... :- effect.

It :1.:.. 's the role of a dielectic insulator between two :.1:::. !. : ., which in this case are

the separate salt solution reservoirs on either side of the membrane. It is necessary

to consider this ,:: ,:. :...' because it ",.. a central role in the determination of the

number of ions that must be transmitted for a current ..I to be observed at the

membrane (Hille, 2001). The < ..: : :: I :':., is defined as the amount of : 1:.:: : that must

move from one reservoir to the other to set up a *.. potential:

C=Q/E

(where Q is the *.... transferred, E is the potential. *. ...... and C is the

i .! .:... .*) The rate of change of potential due to current flow, In, at the membrane is

given by:

dE/dT= Im/C

The capacitance is relatively constant for a .. ::: membrane environment. In terms of

m em brane :: ....: .. ...the : '..::. of their1 ..i .. .... 'is given by the .; ....

C = KEoA/d,

where K is the dielectric constant of the given lipid, Eo is the permeability of free

space, A is the surface area of one of the lipid layers and d is the distance from one

side of the bilayer to the other. The ... ..:.. of cell membranes is estimated to

be ::. ::':: ,:i.:y 1.0 pF/cm 2 and the ,,: :. :..:::..:* for a ::, native! *:, bilayer is

Sh-ttiv less at 0.8 pF/cm2 (Almers 1978). From these measurements, we can estimate

the thickness of a i ,'..1 bilayer to be y: '"::: .... 1:"y23 A, assuming a dielectric constant

for hydrocarbons of 2.1 (Q -.. 2001). The general effect of membrane capacitance is to

slow down the :.'.,; '::" to any current by a time, T, that '. :::. on the resistance and

capacitance of the membrane. Thus we can .- the i : 1.. .. rate of the membrane

,.. :: a s:


dE/dt = I/C = -E/RC = -E/T









Solving this differential equation gives us an expression for the potential across the

membrane as a function of time, t. Given an initial potential, Eo0, this relationship is

E = Eo exp (-t/RC) = Eo exp (-t/x)

The time constants for the different biological membranes and lipid bilayers can vary

significantly, thus the resting membrane resistances can vary in order of magnitude

from 10 to 106 Q (Coronado and Latorre, 1983). Thus the number of ion channels open

at rest varies significantly from membrane to membrane depending on the properties

of the lipids and channels involved. Now we have all of the components to look at the







9i
Cm


E,- ---m
Ei






Figure 4: Circuit Model of a Membrane. The electrical properties
of a membrane can be modeled using the conductance, gi, of ions
crossing the membrane with a membrane potential, Ei in series.
These elements are then in parallel with the capacitance Cm, set up



bilayer/channel environment as a circuit-like model. If we take the ion channel to be a

conductor, with conductance, gi, for a particular ion, i, and the membrane capacitance

and electromotive force of the pore to be Cm and Ei, respectively, then the system can

be shown to behave like the following equivalent circuit in Figure 4. The driving force

of the specific ions, I, is not simply related to E, the potential across the membrane, but







13

instead is determined by the difference of the total potential and the potential at the

pore. Thus the current i::.. of I through the i1:.:::::!. ,/membrane system is *,= =.-..,.... as:

l = gi (E E)

While this is adeq uate to a first :::.. ::: :..= many channels have been observed to

have non-linear current-voltage relationships when open. This non-ohmic behavior can

be due to ionic gradients across the membrane or when the structure of the ion channel

itself lacks symmetry. By looking at current versus voltage (I E) :::*.... a significant

amount of information about the ion channels being observed can be gleaned. For

S if the curves are linear and pass through the origin, then it can be inferred that

either the channels are nonselective if there are multiple salt ions in solution or that

there is no g: .. 1:. :: : set up across the membrane. If the slopes of the I E curves of a

given system :..::.. = :. it corresponds to an increase in the *:: :: '.-alent conductance and

thus an increase in the number of channels in the open state (Hille, 2001). Conductances

that show a dependence on voltage are said to undergo rectification. An environment

that shows strong rectification : : :.: il has channels that are open for some groups of

potentials while remaining closed at others.














CHAPTER 2
i TRIALS AND ".. i i i i )DS


Channel Synthesis

The synthetic oligomeric M2 peptide corresponding to the 255-277 segment
(F-... T->TAISVLLAQAVFLLTSQR) of the 6 form of the T .. 7.', >7 .. AChR was

S.*:: .:::: :. via solid-phase methods. The 6 form was selected due to the fact that it is

well-characterized (Oblatt-Montal et .! 1993, Opella et 1999). Solid-phase chemistry

allows for a much less time-consuming synthesis as isolation and purification is not

required at every step as in solution-phase chemistry. The synthesis was carried out

with PEG-polystyrene resins under continuous flow conditions using FMOC chemistry

by the University of Florida protein core facility. The C-terminal end of the peptide

with the protected first amino acid residue in the sequence is pre-loaded onto the resin.

The next amino acid in the sequence, which was also protected, was activated with

an acid fluoride and added to the F,.. )C-deactivated segment loaded on the resin. A

peptide bond was thusly formed between the two amino acids in the chain. Then the

excess, unreacted amino acids were washed from the resin and the activation and

deprotection steps were repeated with each subsequent amino acid in the sequence

until the full peptide was formed on the resin. A schematic of this .: .. is shown in

Figure 5.

I it was necessary to cleave the N.' 6-subunit peptide sequence from the

resin. The first step in this process was to wash the resin with .. ....: ... 1..- 25mL

DCM for each gram of crude sample resin through a small sintered .. ... funnel. The

resin was then carefully transferred to a round bottom flask where 10mL of cleavage

reagent (90% trifluoroacetic acid (TFA)/ 5% triisopropylsilane/ 5% water) for each

14








IntrducliDn of auino add with Resin pre-l ded wilh first
X2 aldB-ctAn protecting group amino add In asquen:e and
aide-c-ianr pin&c.ng gmrup X-
H H 0 H H 0
I I I| I | I I1
FMOC -N-C -C- OH + Adlastor Piperidlne+ FMOC-N- -C-G-O- )
I I RqRein
R2 R1

X2 x1
Acfvualin ie prtaclion



FMOC-N--C- C -Activator H2N-C-C 0---











I I I
FMOC-N-C-C- HN--------J
t Resin








H H 0 I, Cleavage H 0 H 0 H F

---C-C --C--C--0-( ---HNN -- N-C--- HN--C
I IAmn I II
R2 R1 Rn R2 R1


Figure 5: Schematic of Peptide Synthesis.







16

gram of crude resin sample was added. All spatulas and other transfer tools were

washed into the round bottom flask with the same solution to ensure as high of a yield

as possible. The flask was then 1- .. ....1 and stirred. A small stir bar was ) 1.- *1

inside the flask to ensure :: while the reaction occurs. The solution was allowed to

react for ,. ...... :. ... !y three hours.

The mixture was then filtered : : =:: ': a sintered funnel and was then

washed with ImL TFA. The total filtrate was concentrated under vacuum at 68C to

: ::: ly 2mL. The concentrated solution was slowly transferred into two Falcon

tubes..:: .. : ": ethyl ether at -20C. The solutions were then i:- : ..... d,
y:: 1::. pellets. The ethyl ether was then decanted and the :.... .... was repeated three

times. -..* ..... .: the washes, the ethyl ether was removed under '1.'- vacuum and

then 1 .:.,.. under high vacuum in a desicator for at least two hours.

Channel Purification and Production

The cleaved peptide was purified using an Akta Purifier (Amersham Pharmica Biotech)

reversed .1..: high ?p r-, liquid .-.. .:.. (RP- : ':*:. .. apparatus equipped with

a silica Cig column from Vydac (see Figure 6). This method allows for the separation of

peptides with r: :.:::.:* that. 1 by as little as one methylene :....,. on a single

amino acid (Carr, 2002). Unlike small molecules, peptides are too large to partition

in the hydrophobic j.L .. -: stationary phase. Instead, they must be .:. i ..i .. '. to the

hydrophobic surface of a column. An absorbed peptide remains on the column until

the concentration of .. i organic modifier (:: ,:.: acetonitrile) reaches the c :::, i

value needed for A.' *::: .::: This critical value is know as the Z number and was first
S.... :i .- by Geng and Regnier. The Z number has an extremely narrow region of

concentrations that define it and it is very sensitive to the conformation of the protein

being I: ..i Once desorbed, the peptide interacts very little with the hydrophobic

surface as it elutes off the column. In RP-HPLC, the peptides are !.. .1 based on

slight differences in the "hydrophobic foot" portions of their sequences and structures,









which is the only .. .:.. of the 1.. .' in contact .. :. the hydrophobic surface of the

column. The foot is* : ::..-y correlated with the Z number as it is the only ::-;:: of the

... ;:! that is involved in the interaction with the ..... modifier. In fact, the :-

location of the bf. .l: : : residues in a helical ...*'.1: are :::. ::::, in .:

the retention time of that.:- :. :.-., in the ..... modifier. The .' ........ /desorption

step occurs only once for a .. ::: .:i: .. on the column (Carr, 2002). Since ,: .. "1:"

are very sensitive to the concentration of ... modifier present, elution gradients

are usually set up to maintain ::... ::.:. : ::: The 1'::1. :, :' ..1. are sensitive to

the concentration of.... .....:. modifier used. Relatively small concentration : ....

in the acetonitrile cause large i: .:::: : in the retention time. The : ::: modifier

concentration must be maintained precisely and isocratic elution is difficult due to the

sensitivity of the '::I', ':I : to 1:: : :: :... in the concentration of the modifier solution.

Therefore, for RP-HPLC separations, gradient elution is ... r ..-. even with small

...:.... in : .:::. modifier concentration per unit time. Thus a hybrid ...

method is indicated: small .." ::- .. desorb more quickly with small i .... in modifier

concentration than small molecules, but more slowly than proteins. In a helical 4': :

the exact location of hydrophobic residues .... .' ; influences .... ...... ;. ...'

retention (Yang et al., 1992).

The hy ... .1.1. .-. surface ... which the pol. A.- adsorb is the HPLC

i1::::::: which is a stainless steel tube filled with -. ::,': I: of small diameter.

The particles used are generally silica with -i.r-j.y uniform sites and surface areas,

which have been modified for use as adsorption agents by silane : .-:: .. : ::.. to make

them hy'.- .1.....:1.. The smaller the diameter of the adsorbent j.... :1. the ..

the specificity and resolution of the peptide fractions eluted from the column. The

adsorbents used are porous in nature and the .. 1 .. :.1. 1 enter a pore to be adsorbed

and : ': ... :: .:'.. RP- HPLC has been effectively used for i:: Ii' 'i,';:,. ',' :: : ::: since

ore silica :,. .:'1- of about 300 A were .. .. ed. Now this is the size most









commonly used, but some smaller .'. can also be .- : particles of 100

A (C.--, 2002).

L :::. a linear .: ..: hydrocarbon of 18 (Cls), eight *i or four 1 .: I.

is used as the hydrophobic = P. *!.,. and small proteins of size less than 5,000

daltons are usually separated using Cis columns. In order to :: "-.1:::*.: RP-HPLC

adsorbents, hydrocarbon chlorosilanes with one reactive chlorine are added to the

silica matrix (Carr, 2002). As these have a :*:'.-1- point of attachment with the matrix,

they are called monomerically bonded i.1: : .-. If chlorosilanes -.'. :;1. multiple reactive

chlorines are used, the product is known as a j. 1 t-merically bonded '1:.: .. in which the

individual chlorosilanes are crosslinked and form a silicone }"' Aymer on top of the silica

matrix, .. : multiple 1:::'i .1: chains attached. Monomerically and 5' "..merically

bonded phases exhibit some differences in :.. :': ; ity for ....... ..1 '

even though they have similar hy.L: 1:. *. .. and separation. Mild conditions of

acidic pH and room !.. *,:::== e are ideal for ::= ::= silica-based HPLC but extreme

conditions such as high pH or h.' ::::1 .. ..:: es .. :: ii .: :'.- :' trade silica columns.

Polystyrene-divinylbenzene, a synthetic .. ~ .mer, A.= ides a very sturdy alternative

matrix for separations. Synthetic polymers provide the advantage of not degrading at

extreme 1: ::= in pH. Therefore very acidic or basic solutions can be used to clean

the apparatus. Using very ,!:,:: :. :::. to wash the column does not adversely

the column performance, as indicated by an examination in which -A... '.

chromatographed before and after -..,1:: .. :1:: both strong acid and base had similar
,1 ,: := .. retention, and resolution.

Column: ::...th does not .: :::::: iy the .: .1:.:.. :-:: and resolution of
=.: because both adsorption and I... .. 1 := 'take 1 ..... near the top of the

column (Yang et al., 1992). Short columns (of 5-15 cm) are frequently used for protein

separations. Small peptides are better.- ...= .. .1. on longer columns, and columns of 15-

25 cm are used for the separation of synthetic and natural -:'"1- ., Column: 1::: :" is







19

S......i .....,. to column back-pressure. While the column diameter does not affect peak

resolution, it does ": -*: ::.:. : ..!:,,-. loading. The detection sensitivity is increased when the

column diameter is reduced, because the flow rate is reduced, and therefore less solvent

is used (Rivier et a.l, 1984).

Aqueous solvents contain an ... modifier and an ion-pair .,... I or buffer.

The ::: :: solvent is used to desorb j" b -" '; ;'~=:' molecules from the adsorbent

hydrophobic surface. This is achieved by slowly ..... the concentration of solvent

until the '::,". ; '."".I."" desorb and elute. This results in the --.1'-: :i iest ... and best

resolution. The most commonly used ... modifier is acetonitrile (ACN) for several

reasons: it is very volatile and therefore easily removed from ": .. .::::: : its low' ....

minimizes back-pressure; it has little UV ..1.... at low wa' : ths; it has ....

to be consistently reliable in RP-HPLC separations (Carr 2002, Y.:: :: et al., 1992).

Low-gradient slopes are.. f- to isocratic elution, as a uniform

concentration of. .::::. modifier i, ; : ... -tably broad '. =:1 (Yang et *

1992). The slope of a 1 :21 solvent gradient is 0.5 to 1% per minute increase in -

modifier solution. To maximize resolution, very -. :,:::. -.. gradients :j:: ::::: 0.5 to 0.1% 0:
...:..1'.. can be used. Extremes in ..... .. modifier concentration are to be avoided in

order to have the best equilibration and reproducibility. Gradients should be begun at

no less then 3 to 5% ...... modifer concentration, and should not exceed 95% ..

modifier. The higher concentration of ::: ::.. modifier may remove all the water in

solution, thus .. ..1 .: column ..... more difficult (:r'., 2002).

The slope of the solvent gradient may S=q: = .= '.'.- s.. K.. Iy and resolution

between i .*.. .- because of the slight differences in the way that some of the

peptides interact .. .1: the reversed-phase surface. The purpose of the ion-i: : ..

.1 /buffer is to set the eluent pH and interacts 0.. T:. the pol' :. -: : to enhance the

... ::: (Hoeger et.,. 1987). Trifluoroacetic acid is the most commonly used ion-

....... ...... because it is volatile and can be .,. ..: removed from fractions, it has







20

very little UV adsorption at low wa. 1. ...ths, and it has ........ reliable in RP-HPLC

SU. ::-- concentrations of TFA at lower than 0.1% when chromato'-: :.;'1:

proteins may A.!...ade, .'" .'.*" As the solvent environment. ..... from aqueous

to non-aqueous the dielectric constant, 1B .. : .::. :: n-n electron : ... ...

which affect the 190-250 nm region of the ,..... -'- :. ......., and causes a baseline

shift (Rivier et 1984). Adjusting the we.... .: .:::.. .1: as close to 215 nm and putting 15%

less TFA in the ..... .....: modifier solvent than in the buffer solvent reduces or eliminates

the baseline drift due to TFA .... ::.:1 adsorption. Using high-i ::: TFA is also

essential, as low quality or aged TFA may cause the ... of -... peaks.

Po. :..I':1 .,, :: : ..: *:::. are not very affected by flow rate. The eluent flow

rate may affect the resolution of small peptides because their behavior in RP-HPLC

columns is between that of ,::': :'. and small molecules (Y ::::- et al., 1992, Carr, 2002).

Adsorption and sensitivity increase with lower flow rates because smaller volumes

of solvent are used. This is also why narrowbore HPLC columns increase detection

sensitivity: they are run at low flow rates. However, *...,. it increases the amount

of solvent to be removed from the purified.. 1.:::: 1 ', high-flow rates may : :: the

solubility of hydrophobic pol. .. (Rivier et.:' 1984).

RP-HPLC is used to purify mic ..: to :, ,. : .,,,, quantities of I.'i ;'I.1

for research ** For .,... ...- preparation, however, some other

constraints exist as well (Rivier et al., 1:: .J The same ion- ::. : agents commonly

used for analytical ****j. .... are not always as .. :..' for 1..... ..., chromatography.

When t ..;:::. a ..:: :." "... '::, '.:: ::. some factors that need to be considered are:

low .*. : loads yield best resolution, only small ;:....*::. are purified per run; the

of the ::::,,.. is ... ....1 in terms of ,: :: : ,. of total weight of final purified

product; and the yield is the .... ,..- of final purified product per total amount of

'. : ::. -.:::1n in : : .: :. (Y.: :::- et 1992).Higher resolution enables

maximum yield. Thus several factors must be balanced. The three measures of .,







22
(... ....modifier, and the other between 53-' c......- modifier. 8 mL fractions from

45% to 60% ::: ::::. modifier were collected in Falcon tubes. Then each individual

fraction was placed in a round-bottom =.,. and the ACN and any trace of TFA were

removed using the Roto-Vap under vacuum -.. :i:: a water bath at a .:::,:: ..: ::'e of

approximately 50C and Roto-Vap coolant water at 4C. Once the A. -.... were

concentrated to approximately half their ::: :-;:::.:1 volume, they were removed by glass

-... !!. and put back into the ...... .1 Falcon tubes. For each fraction, the round-

bottom flask was rinsed with deionized water and the water was .'.i --l 'tted into the
... .... .. Falcon tube to ensure maximum yield. If the same round-bottom

flask was used for :1! :. .1,. concentrations, it was rinsed three times ::: ::::: .i;::. with

methanol and deionized water. The ...... x: were then frozen at -30C. Once ..

the : :; '.. of the I:: = :::: tubes were removed, and the tubes were sealed ..,.: 1 :

Then three small puncture holes were made in each parafilm seal and the tubes and

--. w ere : :.:: :! in the glass !- : :: ., : :: container. The ... ::::::, '1: ". w ere 1" :i :

under vacuum at less than 100mTorr until only a white powder remained.

In order to identify which of the :::: ::: :.'.: ". contained the correct ..

the samples were ...... -* for mass ... 1 .. y. First, a solution of ACN with 0.1%

TFA and deionized water ... i1: 0.1% TFA in a one to one volumetric ratio was I::: -: .

A small ..... i*. of the lyophilized *- .. 1 for each major .-1. was dissolved in 90

pL of this solution to yield an -:::: :;: .. concentration of 0.1 pM -. "'!i .1:' Then the

solution was ci: .:: tenfold with the 1:1 ratio of ACN: H20 (both with 0.1% TFA). And

then the diluted fraction was : : ::, diluted with the same solvent. Next a cyano-4-

hydroxycinnamic acid (CHCA) matrix was prepared by dissolving 10 mg sa::::..i- in IX

methanol in 0.5 mL of solvent. The solution was then ..:- .. .- and methanol was then

added until the solution was : :: .:..- 1 Then the matrix solution was centrifuged and

the supernatant was carefully removed. The matrix and the diluted : :: pie were mixed

in a 10:1 ratio. Then 1 of 2 pL of solution for each sample was placed on a specific









location on a MALDI plate. The CHCA matrix was A.... on a few of the MALDI

locations alone for use as a blank. First the blanks were tested to see if good resolution

could be obtained. Then the other samples were tested on the MALDI plate, with the

higher concentration -.,. : :1:" .:. ;:... the best resolution. The results show the ..'

molecular mass of ') amu for the .. -;. collected at .: .:... -. iy 48% .

modifier .1: :::::: the PR-HPLC. The other :.:: i. : :, :1 .. had molecular masses of 2409.38

amu and "'.' ---.31 amu which represented a.. .:.- .... an amino acid residue in the

sequence, most likely valine and 1". : ., '. vely.

To verify the sequence and concentration of the -. :., l-. .1....:: .. samples

of the different ,=. :1 fractions were dissolved in ,: .':: ::.. 1y 100 pL TFE for each 1

mg of sample used. Each .. :.1. was then heated in a water bath at about 40-' .= for

approximately 2 hours. Then the solutions were diluted to 2 mL with H20. Then 100iL

of this dilute solution was further diluted to 1 mL with H20. 100 pL of this solution

was sent to the protein core facility at the University of Florida to have amino acid

analysis performed. Also, this solution was used to obtain UV .. -. 1:.: from 200-400

nm. QuartzUV cells were used because residual p ..1 *. near 200 nm were '.

for blanked solutions when methyl crylate cells were used. The results of the amino

acid analysis were in close .* :'= ::: : for the .1 c. :: ..'..:. .::, of the M 2 :... :i. 1..

The predicted peptide sequence had an average percent error of 12. ::::: .: 1 to

the known ..::..... :..... which is within the error of '.: ."" .* amino acid analysis. The

predicted concentration of the sample was 69.8 -::::,. 1 / pL L or 0.16 pg/pL.

In order to be able to determine concentrations of : solutions with :. : !,-: ease

via UV .:1--:: 1 ..::::: it was necessary to calculate extinction coefficients to be used with

the Lambert-Beer law. The law can be stated as

A= cl

where A is the absorbance, I is the path length, c is the concentration, and is the

extinction coefficient. Extinction coefficients for absorbances at wa.. 1; ::.. ; of 208 nm,









"1 3:.::: and 220nm were calculated: ':== the UV .=.. =., of a known concentration of

M2 in deionized water. This concentration was determined by amino acid analysis

and the path length for the glass cells was 1 cm. Using the Lambert-Beer Law, the

concentration of all subsequent i. ,i ,,1: was determined. The ::-- :: at 208 nm

was determined to be 6.517 x 10-3 Au pL/1::.::. .1 cm or 2.810 Au pL/pg cm. At 214 nm,

the coefficient is 7 .-'" x 10-3 Au pL/pmol cm or 1.638 Au pL/pg cm. And the values

for the coefficient at 220 nm were 2.244x10-3 Au pL/pmol cm and 0.961 Au pL/pg

cm. Once concentration was determined for a particular ::::: 1: it was then :
..:. ... .. and then i ..... :.. ............ up in an equal volume of 30:70 methanol

('.. -..OH): chloroform (CHC13) solution. The synthetic DPhPC l *i *: T were also':.-:: -1-:

up in solution of the same solvent ratio and then solutions of various molar ....

ratios were .::..'.::,.,1 from these stock solutions.

Circular dichroism .. .. ..! were done .." :. .. i. in different solvents to

determine the secondary structure of '.-" in solution. Solutions of the .: :.: =- 1:1

TFE:H20, 30:70 TFA: H20, 30:70 MeOH: H20, 30:70 TFA: CH( i and 30:70 MeOH: CHC13,

each with and without M2 peptide present were prepared. :::::. : pes of 400piL were used

for each run in a cuvette with cell length 0.1 cm. Samples containing chloroform or TFA

scattered the i= =::=! light such that no useful data could be obtained as a clear spectrum

could not be..i ... =. 1 The molar ellipticity as a function of wavelength was plotted

for the M2 in solution, for : :::: pes where ,.: !:= could be obtained. Molar ellipticity

can be ::- as number of millidegrees of rotation of polarized light divided by

the product of the number of amino acid residues in the p-',. !K the concentration of

the peptide, and ten times the cell length. The results of these experiments, after I =

subtraction, showed that M2 was in an unstructured, random coil conformation in

the 30:70 )i'. )H: H20 solution. This is what would be ..- : :1 for a non-aggregating
.............. 1 ; 1. ,F 1... ;. 1., ,
I i '.







25

Monolayer Formation and Isotherm Data Acquisition

after Irwin I :.:: ::::: :, who in the late 1910's was the first to perform

systematic studies on ...- .- monolayers in water, T ......... films are -* .11

in i:-:: :- .......in the scientific community. The name Tl .1A. ett was added several

years later when Katherine Blodgett gave a detailed description of monolayer transfer.

I :- =:::;: films usually refer to floating monolayers, and T :... '-Blodgett films refers

to a monolayer attached to a solid substrate.

The -.!.. ::-:A-- force that causes the formation of monolayers in such a system

is surface tension (Roberts, 1990). Molecules in a liquid i ... ...... attractive forces

in all directions. The molecules on the surface of the liquid :: stronger

intermolecular *....... with other molecules of the liquid as opposed to the air molecules

at the interface. Therefore the liquid will automatically minimize the surface area

1 to the air. F., :. T a minimum surface area exposed to the air also lowers the

of the liquid molecules, which is favorable. The more polar the molecules of

the liquid, the 1....... .. the intermolecular *. ..... involved, and the 1... .... the surface

tension will be. Any contamination will :I. the surface tension, and therefore

researchers should avoid any contamination ...,w, 1980). Also, : .. the

:: :'e will lower the surface tension.

Surfactants are surface active agents and they consist of a 1.. .. .... .1.::- portion

and a t:. .:= =.1:,::.VC, non-polar p:,u: As they are contain segments soluble in

both .. and polar solvents, .. or .. .... molecules tend to

accumulate at interfaces (Laughlin, 1994). Altering the size of both the1:; 1: 1:: 1. and

hydrophobic j- -.. ... ........a wide variety of self-assembled structures, but

all of them come about due to a reduction in free, .. :. .::. .r. T .. :::: :: films are a one-

molecule-thick insoluble monolayer that come about when an ........:. substance is

spread on the surface of water (Roberts, 1990, Binks, 1991). The v ::.': ends stay on the

water side of the interface, and the ...... :olar ends point up. Usually, the hydrocarbon







26

chain, the ....... polar .. ...... must be at least 12 hydrocarbons in order to form a

monolayer rather than a micelle (TT -:::1 :'.. 1952). However, if the hydrocarbon chain is

too long, the molecules .. ::crystallize on the surface rather than f........ .. a monolayer.

Therefore, a delicate balance must be attained.

Water has a very 1.1' surface tension compared to other liquids, and therefore

makes a good subphase for monolayer -. ::.::... The ':: .':::e for f::::::::- a
T..... uir film is as follows: first A *. ... .,. "..- .:. solution (in a non-polar .1. -.0

with micro .. : on the surface of the si ... :,:-.. solution; as solvent dissolves, a

monolayer forms. This monolayer is considered a "gas" because the molecules are

quite far ::.== (Roberts, 1990). Barriers on the far ends of the water trough are ':. :.. I

closer together very slowly. As the surface area of the monolayer decreases, the surface

.. .... increases and the molecules repel one another. The equation

1 = y Yo

for surface pressure, H, is a two-dimensional reckoning of pressure, where y is the

surface tension in absence of a monolayer and yo is surface tension when the monolayer

is A.: .. .

The trough for making a T .:: ::: monolayer is made of Teflon so as to
:. any 1. 1 of the -: '-1. (Ad. .:::: 1976). Water is circulated in channels

underneath the Teflon trough in order to maintain the : '* :: : : ..:..::: The surface

area of the trough can be adjusted with movable barriers. Each barrier is heavy enough

to prevent to prevent any leakage of the monolayer underneath the barriers. The
=:" .. = method is used to measure the surface pressure. A schematic of this

system is ... .. in i :.::::. 7. In this method, a plate is suspended partially immersed

in the subphase. The force exerted by the surface is converted into surface pressure by

accounting for the size of the I".:!. '- (Gaines, 1966, Robert, 1990). The forces on the 1.:

are surface tension ... ::.= downward, ..'. ., ~., and buoyancy .. !:J ... 'i' 1.











by:::- .: ::::'::the surface pressure as the : area is decreased by ::: of

the monolayer. Sometimes, many repetitions are:, ..:': .1 to find isotherms that are

:. .: .::: 1*1- as small variations in the system variable or the presence of contaminants

can have a ': f: ... : effect on the monolayer behavior. Several areas of constant

slope become evident upon examination of an isotherm. The :.- .."::: are known as

S. Generally, the :11 .. behavior is determined by the physical and chemical
.* : .: of the .::::: -1:: 1 the -.:::1i 1:.:..- com position, and .. ::: : .:. :: 'e (Roberts,

1990, Binks, 1991). The !i. of the hydrocarbon chain and the magnitude of any

other forces existing between the head. :..: :*. determines the various monolayer

states. The surface-pressure-area isotherm can be condensed by ::: .. :: the chain

.. ::.. !:, which in turn increases attraction between molecules. A nomenclature system

was i,.. '-'L; -""1 by W.D. Harkins in which monolayers in the gaseous state are given

names (G) (Roberts, 1990). When compressed they will transition to the (L ) state

known as the liquid-expanded state. When compressed further the liquid transitions

to the liquid-condensed state (L2) and when condensed further it becomes the solid

state (S). However, if the monolayer is compressed any further it will collapse. The

collapse is evidenced by a rapid decrease in surface pressure or a break in the isotherm.

Several other critical points exist in the surface-pressure-area isotherm, such as A.

which is .::::::, -::. by the pronounced increase in the surface pressure as well as surface

pressures at which phase transitions occur between the other states, marked by a

1::. ::.: in slope.

The ... :... behavior of M2 and DPhPC was studied T. .. T .. uir

monolayers as described above. Surface-pressure-area isotherm data was- ....:: :: '..

.. a KSV 5000T ..... uir ...... ..1 with dimensions 44 cm x 7.35 cm and a: ......

balance (see Figure 8). The 30:70 MeOH: CHC13. i::, .:: :.:::. solution ,::... ::::.1









Brewster Angle Microscopy

When looking at the angle of incidence of non-polarized light on a flat insulating

surface, at one specific angle all of the reflected light waves are polarized into a single



SMonolayer


| Supphase


Figure 9: Principle of Brewster Angle Microscopy. When incident light
reaches the subphase interface (left) it is transmitted and refracted toward the
membrane normal. However, the addition of a monolayer at the air-water
interface allows for reflected, polarized light at the specific Brewster Angle.


plane. This angle is known as Brewster's angle. It can be calculated using the following

equation:

n = sin(Oi)/sin(O,) = sin(Oi)/sin(O9o-i) = tan(Oi)

where n is the refractive index of the medium from which the light is reflected, 61 is the

angle of incidence, and 0r is the angle of refraction (see Figure 9). It is therefore

very easy to calculate the refractive index of an unknown substance. Snell's law is

inapplicable for opaque materials with a high absorption for transmitted light, so


figure 1I: Brewster Angle Microscope.







31

this 1. is ... i...I Also, this technique determines the amount

of,. I :::: :,.::, and can be used to establish what :: -1:: : the monolayer is in. The

.... used is shown in : -. .. 10. **. ..- / ... Micros. *.. was used in

S ::: with the I.::::::::: '-Blodgett conditions c',.. =:= i above in order to get a

:.'.. .... ..'e of the morphologies of the mixtures at the interface.

Patch Clamp Recordings

The patch: 1. *. method for 1.. .... .. ,-channel activity was first developed in

1976 by Neher and Sakmann. The ; *:'..1:: refers to either a full or p.::,:1 attachment of

membrane or lipid .:. *." .. to the tip of a .. .... that encases a microelectrode that

can be used for .I: .= .. .' small electrical ......... There are two *.* p,. of. I,::: :-.. that can

be .,: -.' :1 to cell membrane or lipid *... ..... The first is .. .....a current

through a membrane 1'.= "= and =1 ..= ::: the potential difference across the membrane.

However, more commonly used for smaller systems is a voltage i... :;, in which a

defined :. 1.'::I'.=1 difference is ,= .. ,1 across the membrane and the corresponding

current flow is measured. What is essentially created is an equivalent circuit whose

components are made up of the : -:' membrane, the::::: ...... ::: -. and the ,:::1:

solution. This equivalent circuit is shown in Y: .. .. 11. In order to observe channel

function .,. : ,i1:::::: the iT ',.1 environment, a .: :. ":: seal must be made that ..- '.:: ..... the

solution in the micropipette, which acts as a conductor, and the .. .** (I-. 2002).

This can be done either by t': = *::. = the::: pipette tip ::, :'= :,. the membrane of a

vesicle or a whole cell or by < ..... a !..1'. ... at the tip :",' a method known as

tip- .. :::. As was discussed in the introduction.,. ::: : :::" the electrical .:::: ::' ..

of .... .. the resistance observed at the tip depends on the resistance of the I'- :.

layer itself as well as on the .:l: :. ive effect of the membrane. The tip resistance is also

affected by leak .. .... which reduces the effective resistance of the patch as well

as the :'.:. 've effect of the glass p'."',. Single channel measurements were made

: the tip-O:K.- patch 1. .. method (Coronado and Latorre, 1983, Suarez-Isla,









Wan, et al, 1983). First, the micropipettes used to house the electrode were prepared

using a Flamming/Brown Micropipette Puller (Model 97, Sutter Instruments Co.) with

a heated filament. The micropipettes were pulled to defined micron-scale apertures of

approximately 1 pm2, as verified by bubble-rate test. Tip-dipping experiments were

performing in a circular Teflon trough that could be manipulated vertically. Also,

some experiments were done using a three-axis manual micro-manipulator. The

subphase solution used was composed of 500 mM KC1, 5 mM HEPES, and 1 mM CaC12,

made with milli-O water and brought to DH 7.4. Bilavers of DPhPC with incorDorated
Equivalent Circuit for an Excised Patch Configuration


Palcl Pipotte l0letrode-


R_ 11 m_ nm 0 .
CFmem


v Patch


Figure 11: Excised Patch Configuration. The electrical properties of the micropipette and the
membrane must be considered when making patch recordings. The membrane resistance, which
is important for determining the presence of a seal at the micropipette tip, can be expressed in
terms of the membrane capacitance and the time constant, Tmem, of the bilayer. Adjustments are
marked at the amplifier to counteract the effect of pipette resistance and capacitance.







37

This c........ ...... to ..... : ... .y 2 to 4 times the amount necessary to cover the

surface area of the .::: 1: .:*. in the Teflon trough exactly, to ensure that at least a single

monolayer covered the entire surface. It is estimated at these concentrations that there

are on the order of tens to hundreds of single channels present in any .. :::. tip.
















CHAPTER 3
RESULTS AND DISCUSSION


First let us look at surface-pressure-area isotherms of pure M26 peptide. As it is

amphipathic, we might predict that its behavior would be comparable to that of other

amphipathic molecules such as lipids. Looking at the isotherms, we see that they have a

shape that supports this proposition. Three distinct kinks occur in the ( ::: indicating

that the peptides on the surface are present in three separate phases before collapsing.

The area per molecule at these different phases can be estimated by taking the slopes

of the curve at each of the phase transitions and examining where the slopes intersect

with the x-axis, ..,. i:::. .:: measures area per molecule. From these data we are able to

determine area of onset. The onset area is the area per molecule at the beginning of a

1 '.:: : :: i.: phase. The limitation area is the area per molecule just before the next phase

transition. Figure 16 0 .; .. !. the phase transitions for M2 :: 1:. 1 at a concentration of

0.0625 mg/mL in 30:70 MeOH: Chloroform. Under these conditions, the area of onset

is 268 A2 and the film remains in this first phase, which is analogous to a gas I'...'..

(G). Once the surface pressure is increased to about 5.9 :::' ./m, we see transition to

the next phase, termed the liquid .... .. l.. .. (Li). Again, the area of this phase

is determined by the slope of the curve, following the corresponding phase transition

.... This area was determined to be 194 A2 and remained in this phase between

surface pressures 6.7 and 28 1::T "/m. The final phase is present above 30 mN/m until

S..^....." at.~ ..... ...i. y 40 m N/m Ithas an area of 136 A2, ;...... : ....to







45
leaky seals or formation of non-gating oligomer pores by M2. In order to show this a

gradient was set up across the membrane formed at the tip of the patch clamp pipette.

With no applied voltage across the membrane, the baseline remained flat, as shown

in Figure 22. When a voltage was applied, single-channel fluctuations could be seen,
OmV
T p








IPAL

10 s

Figure 22: Single-Channel Recording of 1:1000 M2:DPhPC with
Ionic Gradient at 0 mV When no voltage is applied across the
membrane, we see no change in the observed current, indicating that

confirming that the pentamer was forming, and functioning as a voltage-gated channel

as seen in Figure 23.

Once this was confirmed, we could continue with other electrophysiology

experiments. Experiments were carried out with the ion gradient across the membrane
-140 mV
















the bilayer and are functioning as voltage-gated ion channels.
the bilayer and are functioning as voltage-gated ion channels.







50

pharmac.-. response of the conductance, mean ... time, and A.. ... *.. abilitiess in

the ..:::: of agonists, such as anesthetics (T,::::.. and Xu, 2002).

Other future work that should be considered includes .i ...:.... surface-

pressure-area isotherms for the native lipids with and without cholesterol. This would

allow for a more systematic comparison of the .. ...* ..i of the lipid environment in the

::..,.: of M2 peptide. Also, structural studies .:..::: solid-state NMR techniques

should be considered in order to better understand the dynamics of the channel in these

different environments. Such studies may help to: :,i:;: the observed increase in

mean -. :... time as the lipid environment increased in -. .-; -. ..,.















APPENDIX

RAW DATA


The data presented in this .: *. ::. 1:: are those used for the computation of the

values in Table 2 of the text for mean conductance, mean open time, and mean open

probabilities for the DPhPC lipid environments (9 total traces). A trace is shown for

each of the other lipid environments and more raw data can be obtained upon request.

The estimates for the gating voltages were made from data not presented here. In most

cases, the data include the filename for each data set as it i in the compiled da-

tabase, the recordings after baseline adjustment and filtering at I kHz with a Bessel

8-pole filter, and a plot of the open versus closed probabilities for each recording. Note

that in some cases there is no open versus closed graph i=: i.::: i. i. for :.. I: :::..:: data sets.

This is due to the fact that plots could not be obtained that were consistent with the raw

data, however in these cases software calculations that were consistent were obtained

and were used in calculations.














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BIOGRAPHICAL "iiCH


Frank Raucci was born in Pittsburgh, Pennsylv ..... in 1981. His family moved

to T::: U. :-Tequesta, HT. : :. :: where he spent the::: :j::: : :y of his childhood, in 1984. He

attended Suncoast Community High School in Riviera Beach and graduated in 1999

from the .. !:?1:, S :.=* and T: :,=.., :.:" and International Baccalaureate '.. .-:::: t Pro-

grams. His formal, : ............ .*'y education .. at the University of Florida in the

Fall term of 1999. While ::: =. *: -.:! ::. in undergraduate research projects throughout

his time at the University, he graduated .... ...., cum laude in 2003 with a Bachelor of

Arts degree in physics and a Bachelor of Science degree in chemical ::. ::": :,: ::-: He

then 1. .... work on this project as a '*-.: *.- *. *1 for his Master of Science degree in bio-

medical .::" :,,: :::* Frank .. .= be i::-=::." the Medical College of V ':":' at V.":::

ia Commonwealth U ..;. : in Richmond. He ,. be ...'. -:.:.i ..: in the MD/PhD

pro". :', := at this institution.