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KINETICS AND MECHANISM OF MEMBRANE INTERACTIONS WITH
ANTIMICROBIAL PEPTIDE ANALOGS OF CECROPIN A AND MAGAININ 2
Kim S. Clark
A Thesis Submitted to the
University of North Carolina Wilmington in Partial Fulfillment
of the Requirements for the Degree of
Master of Science
Department of Chemistry and Biochemistry
University of North Carolina Wilmington
2010
Approved by
Advisory Committee
Richard M. Dillaman S. Bart Jones
Paulo F. Almeida
Chair
Accepted by
Dean, Graduate School
ii
This thesis has been prepared in the style and format
consistent with the journal
Biochemistry
iii
TABLE OF CONTENTS
ABSTRACT .....................................................................................................................................v
ACKNOWLEDGEMENTS ........................................................................................................... vi
DEDICATION .............................................................................................................................. vii
LIST OF TABLES ....................................................................................................................... viii
LIST OF FIGURES ....................................................................................................................... ix
INTRODUCTION ...........................................................................................................................1
MATERIALS AND METHODS ...................................................................................................13
Chemicals ...........................................................................................................................13
Peptides ..............................................................................................................................15
Buffer Preparation ..............................................................................................................15
Large Unilamellar Vesicle Preparation ..............................................................................16
Lipid Concentration Determination ...................................................................................16
7-Methoxycoumarin Lipid Probe Synthesis ......................................................................17
Fluorescence Experiments .................................................................................................18
Circular Dichroism Experiments .......................................................................................23
RESULTS ......................................................................................................................................24
Kinetic Binding Experiments .............................................................................................24
Dye Efflux Experiments ....................................................................................................36
ANTS/DPX Assay .............................................................................................................41
Circular Dichroism Experiments .......................................................................................45
Thermodynamics of Peptide-Lipid Interactions ................................................................48
iv
DISCUSSION ................................................................................................................................51
FUTURE WORK ...........................................................................................................................55
ABBREVIATIONS .......................................................................................................................56
REFERENCES ..............................................................................................................................57
v
ABSTRACT
There are many factors which control the interactions of a peptide and membrane. The
kinetics and mechanism of these interactions were investigated for variants of cecropin A and
magainin 2. In these variants, amino acid residues were mutated in an attempt to conserve the
properties of the parent peptide while minimizing variety in the variant peptide’s sequence. Cell
membranes were modeled with large unilamellar vesicles composed of various neutral and
negatively charged diacyl phospholipids. Fluorescence experiments were performed to measure
binding kinetics and dye efflux, and the ANTS/DPX assay was used to determine the membrane
perturbation mechanism. Peptide helicity was analyzed using circular dichroism. The binding
kinetics were in line with our estimates, and the efflux levels of the parent peptide and the variant
peptides were the same. However, the mechanism of membrane perturbation changed in both
cases, from all-or-none in the parent peptides, to graded in the variants.
vi
ACKNOWLEDGEMENTS
I would like to extend my sincere gratitude to Dr. Paulo Almeida, whose support, advice, and
guidance made this work possible. Muito obrigado.
I would like to thank Dr. Antje Almeida, whose insight and perspective helped shape mine and
open my eyes to the different view of stuff. Vielen Dank.
I would like to thank Dr. S. Bart Jones and Dr. Richard Dillaman for their patience and feedback
on my advisory committee.
I would like to thank Laura Huskins, Sterling Wheaten, Erin Kilelee, Sarah Higgins, Jeff Naro,
Alicia McKeown, Alex Kreutzberger, Sarah Pagentine, and Julia Nepper for their assistance in
techniques, letting me bounce ideas off of them, washing dishes, for being such great labmates,
and making my time in the Almeida lab thoroughly enjoyable.
The Department of Chemistry, the United States Army Reserve and the National Institutes of
Health provided financial support for my research and studies.
vii
DEDICATION
I would like to dedicate this thesis to my wife: Sherri, you are the love of my life and the reason
I live.
viii
LIST OF TABLES
Table Page
1. One-Letter Amino Acid Sequences of the Native and Modified Peptides ..........................5
2. Cecropin A and CE2 On- and Off-Rate Constants and
Equilibrium Dissociation Constants ..................................................................................30
3. Magainin 2 and MG2 On- and Off-Rate Constants and
Equilibrium Dissociation Constants ..................................................................................35
4. Thermodynamic Parameters for Peptide Binding and Insertion into POPC
Bilayers at Room Temperature ..........................................................................................50
ix
LIST OF FIGURES
Figure Page
1. Major Structural Classes of Antimicrobial Peptides ............................................................4
2. The All-or-None and Graded Release Mechanisms ............................................................7
3. Helical Wheel Projections of Cecropin A and CE2 Peptides at Neutral pH .......................9
4. Helical Wheel Projections of Magainin 2 and MG2 Peptides at Neutral pH ....................11
5. Structure of POPC, POPG, and POPE ...............................................................................14
6. Absorption and Emission Spectra of Tryptophan and 7-Methoxycoumarin .....................19
7. Schematic of Fluorescence Resonance Energy Transfer from Tryptophan to
7-Methoxycoumarin ...........................................................................................................20
8. Lipid Concentration Effects on Peptide Binding ...............................................................25
9. Kinetics of CE2 Binding to Various Compositions of POPC/POPG Vesicles ..................27
10. CE2 On- and Off-Rates as a Function of POPC Content in Mixed
POPC/POPG Vesicles ........................................................................................................28
11. CE2 Equilibrium Dissociation Constants as a Function of POPC
Content in POPC/POPG Mixed Vesicles ..........................................................................29
12. Kinetics of MG2 Binding to Various Compositions of POPC/POPG Vesicles ................32
13. MG2 On- and Off-Rates as a Function of POPC Content in Mixed
POPC/POPG Vesicles ........................................................................................................33
14. MG2 Equilibrium Dissociation Constant as a Function of POPC
Content in POPC/POPG Mixed Vesicles ..........................................................................34
15. CE2 Dye Efflux Experiments at Various Concentrations of 50:50
POPC/POPG Vesicles ........................................................................................................37
16. MG2 Dye Efflux Experiments at Various Concentrations of 50:50
and 70:30 POPC/POPG Vesicles .......................................................................................39
17. MG2 Dye Efflux Experiment with 50 µM 100% POPC Vesicles .....................................40
18. ANTS/DPX Assay for CE2 ...............................................................................................43
x
19. ANTS/DPX Assay for MG2 ..............................................................................................44
20. CD Spectra for CE2 with 50:50 POPC/POPG and 100% POPC.......................................46
21. CD Spectra for MG2 with 50:50 POPC/POPG and 100% POPC .....................................47
INTRODUCTION
There has been a vast amount of research on antimicrobial peptides since their discovery
three decades ago (1). These are small, endogenous peptides characterized by their ability to
bind and neutralize various infectious microorganisms (2). Antimicrobial peptides have an
uncanny ability to efficiently discriminate between self and non-self which reasonably justifies
their presence in all kingdoms of life (3). The means by which they defeat foreign cellular
bodies arises from the nature of the peptides themselves. These peptides in general contain
between 10 and 50 amino acids, are basic and positively charged at biological pH, and are
amphipathic. These peptides vary in several properties which contribute to their unique
interactions with cell membranes, and hence their activity: sequence, size, structure, charge,
hydrophobicity, and amphipathicity (4). Variability in these characteristics generates several
results: it allows an organism to produce peptides which can respond to an assortment of
different invading microbes based on their cell type and allows several peptides to work
synergistically to overcome infection by departmentalizing their functions. The latter may take
place because some peptides function by direct interaction with and disruption of the membrane.
Others permeate the membrane of a foreign microorganism and interact with internal
components of that cellular system (5).
The sequence of the peptides determines the interactions that it will have with potential
targets as well as what secondary conformation the peptide can adopt. In turn, the secondary
structure determines how the amino acid residues arrange themselves spatially, contributing to
hydrophobicity and amphipathicity. The hydrophobic character determines how easily the
peptide can partition from the biological matrix to the membrane. The amphipathicity is the
arrangement of the nonpolar hydrophobic residues opposite from positively charged basic
2
residues. Many peptides adopt this arrangement upon interacting with a target membrane (6).
The size of the peptides determines their area and thus defines the potential facial interaction, as
well as other physical properties of the peptide such as overall charge and charge distribution.
As with all biological systems, there is a dynamic relationship between a peptide, its
biological transport system (solvent), and its specified target (membrane surface). Most
eukaryotic organisms’ primary defense against bacteria, fungi, and viruses is attributed to
cationic antimicrobial peptides (5,7). Current work also probes the feasibility of using certain
peptides as anticancer treatments (8).
As the first tier defense, these peptides must be nonspecific for invading bodies and either
eliminate the infection or provide sufficient time for the host adaptive immune system to be
mobilized. With these requirements, these peptides are often found in the epithelial layers and
within phagocytic cells in larger organisms (9). The short sequences of amino acids facilitate
simple and rapid synthesis, whether the assembly machinery is biological, in vivo, or synthetic,
in vitro (4). The evolutionary success of antimicrobial peptides is demonstrated in a cell’s ability
to respond quickly to infectious agents and evidenced by their occurrence in a variety of species.
With the increase in resistance due to widespread misuse and overuse of current classical
antibiotic treatments, alternate means of treating infections must be explored.
Though antimicrobial peptides are sequentially, synthetically, and structurally simpler
than proteins, the exact correlation between structure, function, and activity is yet elusive; there
probably is no direct correlation between them (10). It is our hope that this research will shed
some light in this area. This study attempts to reinforce established relationships between these
properties to better understand how various antimicrobial peptides affect permeabilization and
3
liquidity of cells. More precisely, we hope to show that the specific amino acid sequence is not
important by itself, but that the properties of the amino acids at specific locations in the primary
sequence affects overall peptide action. The kinetics and mode of action of peptide-lipid
interactions were compared to the native peptide by modifying the primary sequence and
replacing several amino acid residues with a ―minimalist‖ analog. It was our intent to preserve
the secondary structure of the antimicrobial peptides, and gauge the effects caused by
conservatively modifying targeted individual residues.
There are several structural classes of antimicrobial peptides. The major structure types
include α-helical, β-stranded, extended coil, and loops (11). Examples of these can be seen in
Figure 1. The peptides in this study are solely the α-helical type: cecropin A, and magainin 2.
We wanted to test the hypothesis that mutations of most residues in a peptide are neutral,
attributing minimal change to the overall peptide properties. In our studies, these two peptides
were modified into WAL analogs, composed of a tryptophan, and many alanine and leucine
residues. These are ―minimalist‖ versions of the peptides where Leu (L) replaced all
hydrophobic residues and Ala (A) replaced all others. The intrinsic fluorophore Trp (W) was
retained when included in the original sequence, or was added. The residues Gly, Pro, Asp, Glu,
and Lys were maintained to conserve charge, except that Arg was replaced by Lys. Some
additional residues were modified to either retain conformational requirements of the peptide
sequence, or to keep thermodynamic values close to the values of the native peptides. The major
differences between the peptides arise in the kinetics of peptide-membrane interaction, the
energy required for insertion into the lipid bilayer, and the efflux kinetics of lipid contents. We
expected that the neutrality of the mutations would produce results that are indistinguishable
between the native and WAL mutant of the peptides, the detailed sequences of which are shown
in Table 1. The cecropin A mutant is called CE2 and the magainin 2 mutant is MG2.
4
Figure 1: Major structural classes of antimicrobial peptides. The yellow colored ribbons represent β-sheets, and the
magenta colored portions in (C) represents an α-helix and in (D) represents the loop portion. (A) Extended
indolicidin (PDB ID 1G89); (B) β-stranded hepcidin (PDB ID 1M4E); (C) α-helical magainin 2 (PDB ID MAG2);
(D) looped thanatin (PDB ID 8TFV). These were modeled using the RasMol molecular graphics program, v.2.7.5
(12).
C
A B
D
5
Table 1: One-Letter Amino Acid Sequences of the Native and Modified Peptides. The gray boxes highlight
mutations from the original sequence to the variant.
Peptide Sequence
Cecropin A KWKLF KKIEK VGQNI RDGII KAGPA VAVVG QATQI AK-amide
CE2 KWKLL KKLEK AGAAL KEGLL KAGPA LALLG AAAAL AK-amide
Magainin 2 GIGKF LHSAK KFGKA FVGEI MNS
MG2 GLGKL LHAAK KLGKA WLGEL LAA
6
The means by which the antimicrobial peptides perturb a membrane can vary, but can be
classified as either all-or-none or graded. More popular mechanisms include the barrel-stave
model, the toroidal pore model, the carpet model, the sinking raft model, and other models
describing less structured pores (13). In the all-or-none model, individual vesicles either
completely release their contents or they release nothing at all. In the graded model, all vesicles
release the same portion of their contents. A simplified diagram representing the two extremes
of the all-or-none and graded mechanisms of release is shown in Figure 2.
The all-or-none release mechanism is normally attributed to the barrel-stave model, the
carpet model, and in some instances, the toroidal pore. In this mechanism, the Gibbs free energy
of insertion of the peptide from the membrane interface to the hydrophobic core of the bilayer is
hypothesized to be larger than 20 kcal/mol (10). This mechanism consists of four different
states: 1) unstructured, unbound peptide free in solutions; 2) peptide bound as an α-helix to full
vesicles; 3) peptide inserted into the lipid bilayer in a pore-state; and 4) peptide bound to empty
vesicles (14).
The following are several models which display the necessary criteria for the all-or-none
mechanism. In the barrel-stave model, several peptides insert into the membrane perpendicular
to the bilayer forming a pore—the peptides, in contact with each other, form the ―staves‖ and the
overall structure provides the ―barrel‖ shape (15). The hydrophobic portions of the peptides
point toward the acyl chains of the lipids and the hydrophilic regions line the solution side of the
pore, allowing cytoplasmic contents to easily cross to the extracellular region and also causing
potential loss of electrochemical gradients. The carpet model involves peptides orienting
themselves parallel to the lipid bilayer, and upon reaching a critical threshold, they cause
permeabilization (6,9). They do this by coating the membrane like a carpet, which requires
7
Figure 2: The all-or-none and graded release mechanisms. All-or-none release of 50% of the vesicles causes half of
the vesicles to release all of their contents and the other half to remain fully intact. Graded release of 50% of the
vesicles contents causes all vesicles to lose half of their contents. The details of the different mechanisms were
omitted from this schematic drawing, but are discussed in detail by Yandek et al. (14).
8
relatively high concentrations of peptides compared to other models. Since the concentration of
peptides in this model is high, the action of the peptides is detergent-like and can cause
micellization. The toroidal pore model (16,17) involves several peptides inserting
perpendicularly into the membrane, however, unlike the barrel-stave model, they do not
necessarily have to be in contact with each other. Portions of the membrane can fold in and fill
the space between the peptides, creating peptide and lipid lined pores.
Graded release is attributed to the toroidal pore and sinking raft mechanisms of
antibacterial peptide action. However, for toroidal pores to invoke graded release, the pore state
must be short-lived. In the sinking raft model, a stochastic structure of peptides complex and
form the pore (18-20). The structure formed in this model can have the peptides insert into the
membrane either perpendicular, parallel, or both. The mechanism of release is determined by the
type of peptide as well as the free energy of insertion. It is our working hypothesis that if the
free energy of insertion is greater than 20 kcal/mol, then the release will be all-or-none, and if it
is below this threshold, it will be graded.
Cecropin A is a 37 amino acid peptide derived from the giant silk moth, Hyalophora
cecropia (21,22). Upon binding, cecropin A adopts a secondary structure with two alpha-helical
regions, one from Phe5 to Lys21 and the other from Pro24 to Gln37, linked by a Gly-Pro break
(22,23). These two regions can be seen in a helical wheel projection in Figure 3. In this
projection it is easy to see the amphipathicity that the peptide can adopt as a helix. Also visible
in comparing the native and minimalist WAL versions of the peptide is the conservation of the
residue properties at each location. The only noted change in individual amino acid properties is
seen at position 11: the mutation Ala11 Val11, going from an uncharged polar residue to a
nonpolar residue.
9
Figure 3: Helical wheel projections of cecropin A and CE2 peptides at neutral pH. A and B are projections of
cecropin A. C and D are projections of CE2. A and C represent the first helical segments of the peptide, composed
of residues 5 to 21. B and D represent the second helical segments, residues 24 to 37. Blue symbols are basic,
positively charged residues. Red symbols are acidic, negatively charged residues. White symbols are polar but
uncharged residues. Gray symbols are nonpolar residues.
C A
D B
10
Magainin 2 is a 23 residue peptide originally extracted from the skin of the African
clawed tree frog, Xenopus laevis. When discovered, it was aptly named from the Hebrew word,
―magain‖, meaning ―shield‖ (24). Magainin 2 adopts a single helix conformation upon binding.
The helical wheel projections of Magainin 2 and MG2 can be seen in Figure 4. Experiments
show that overall, magainin 2 invokes all-or-none release with data that suggests the mechanism
is either toroidal pore formation or the sinking raft model (25).
Large unilamellar vesicles (LUVs) were used to model bacterial cell membranes in these
experiments. For some experiments, incorporation of a fluorophore was necessary. The
concentrations of diacylphosphatidylglycerol (POPG) and diacylphosphatidylcholine (POPC)
lipids were varied based on previous work with the respective peptides to test the similarity of
effectiveness of the WAL mutants and the native peptides. These model the polar head groups
of lipids found in microorganisms – phosphatidylglycerol being anionic and phosphatidylcholine
being neutral. Varying the compositions of the lipid vesicles allows production of LUVs which
have cell membrane characteristics, simulating native peptide binding conditions.
The kinetics of peptide binding will be calculated for the WAL mutants and compared to
the native peptides using fluorescence energy transfer experiments. Each peptide being studied
has a Trp residue which is used to transfer energy upon excitation to a fluorophore, 7-
methoxycoumarin (7MC), which is incorporated into the lipid vesicles by attachment to the
phosphate headgroup of the lipid diacylphosphatidylethanolamine (POPE). The 7MC is a probe
that indicates the proximity of the peptide to the vesicle. The neutral residue mutations that we
are investigating should impart little or no change in the binding constants previously determined
for the native peptides.
11
Figure 4: Helical wheel projections of magainin 2 (A) and MG2 (B) at neutral pH. The helical portion of these
peptides includes the entire sequence. Blue symbols are basic, positively charged residues. Red symbols are acidic,
negatively charged residues. White symbols are polar but uncharged residues. Gray symbols are nonpolar residues.
B
A
12
MG2 has a calculated Gibbs free energy of insertion of 25 kcal/mol, based on an
estimated helicity of 70%. We expected that this peptide should release vesicle contents by an
all-or-none mechanism. Since CE2 has calculated insertion energy of 36 kcal/mol with an
assumed helicity of 70%, all-or-none release was also expected with this peptide. One caveat to
consider is the helicity of the peptide in free solution and the helicity of the peptide in the bound
state. In the original hypothesis proposal, calculations were performed using an assumed helicity
of 70% in the bound state and minimal helicity free in solution. However, the experimentally
determined helicities are different than this and affect the values obtained for the free energy of
insertion and the thermodynamic values calculated previously.
Incorporation of a fluorescent dye within the LUVs facilitates studies of the efflux
kinetics of the vesicle in the presence of peptide. Initially at self-quenching concentrations
(50mM), carboxyfluoroscein dye cannot fluoresce while still encapsulated in the LUV. Once the
membrane is perturbed, dye released into external buffer is diluted and can fluoresce. This
allows kinetics and total release levels to be calculated.
13
MATERIALS AND METHODS
Chemicals
1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-
glycero-3-phospho-(1’-rac-glycerol)(sodium salt) (POPG), and 1-palmitoyl-2-oleoyl-sn-glycero-
3-phosphoethanolamine (POPE) were all purchased in chloroform solution from Avanti Polar
Lipids, Inc. (Alabaster, AL) (see Figure 5). 7-methoxycoumarin-3-carboxylic acid, succinimidyl
ester (7MC), 5-(and -6)-carboxyfluorescein (CF), 8-aminonaphthalene-1,3,6-trisulfonic acid,
(ANTS), and p-xylene-bis-pyridinium bromide (DPX) were purchased from Molecular
Probes/invitrogen™ (Eugene, OR). 3-morpholinopropane-1-sulfonic acid (MOPS), ULTROL®
grade, was purchased from EMD Chemicals (Gibbstown, NJ). Potassium chloride (KCl),
potassium hydroxide (KOH), ethylenediaminetetraacetic acid (EDTA), and sodium azide (NaN3)
were all purchased from BDH (West Chester, PA). Ethanol (EtOH), 200 proof, was purchased
from AAPER Alcohol and Chemical (Shelbyville, KY). Dichloromethane (DCM), methanol
(MeOH), and all other organic solvents were HPLC or ACS grade and purchased from Burdick
& Jackson (Muskegon, MI). Ammonium molybdate, ACS grade, was purchased from Thermo
Fischer Scientific (Fairlawn, NJ). Dimethylformamide (DMF), ascorbic acid (USP grade) and
perchloric acid (70%, ACS grade), were purchased from Mallinckrodt Chemicals (Phillipsburg,
NJ). Water, filtered to 18.2 MΩ∙cm purity using a Milli-Q Direct Water Purification System by
Millipore (Billerica, MA), was obtained on site and stored in a 20 L Nalgene® carboy.
14
Figure 5: Structure of POPC, POPG, and POPE. The only difference between these phospholipids appears in the
headgroup. Neutral, zwitterionic POPC is shown (top). Negatively charged POPG is shown as a sodium salt
(middle). Neutral POPE, a reactant in the 7MC-POPE synthesis is also shown (bottom).
15
Peptides
CE2 (KWKLLKKLEKAGAALKEGLLKAGPALALLGAAAALAK-amide) lot:
B05973, >82% purity, was purchased from Bachem, Inc. (Torrance, CA). CE2, lot: pr1770, 98%
purity, and MG2 (GLGKLLHAAKKLGKAWLGELLAA), lot: pr1202, 94% purity, were
purchased from New England Peptide, LLC (Gardner, MA). Lyophilized peptide was stored at
-20°C. Stock peptide solutions were prepared by mixing lyophilized peptide in 1:1 (v/v)
water/ethanol. Peptide concentration was determined using a Cary 1E UV-Vis
spectrophotometer, in matched 1.000 cm 6Q quartz cells, scanning from 500 to 250 nm using the
absorbance maximum of tryptophan at 280 nm with an extinction coefficient of 5,579 M-1
cm-1
.
Solutions were then aliquoted into small Eppendorf™ tubes and flash frozen using either liquid
N2 or an acetone/dry ice bath. Peptide solutions were stored at -80°C and kept on ice during
experiments.
Buffer Preparation
MOPS buffer was prepared in water with 20mM MOPS, 100 mM KCl, 0.1 mM EDTA
and 0.02% NaN3, then brought to pH 7.50 using 1 M KOH and brought to final volume. CF
buffer was prepared by grinding CF powder with mortar and pestle, then mixing 50 mM CF, 20
mM MOPS, 0.1 mM EDTA, and 0.02% NaN3, in water and brought to pH 7.50 with 1 M KOH,
then brought to final volume. ANTS/DPX hydration buffer was prepared in water with 5mM
ANTS, 10mM DPX, 20mM MOPS, 80mM KCL, 1.0 mM EDTA, and 0.02% NaN3.
ANTS/DPX titration buffer was prepared in water with 45 mM DPX, 20 mM MOPS, 30 mM
KCl, 1.0 mM EDTA, and 0.02% NaN3.
16
Large Unilamellar Vesicle Preparation
All glassware and syringes were cleaned by rinsing or vortexing with 1:1 DCM/MeOH.
Large unilamellar vesicles (LUVs) were prepared by combining appropriate volumes of stock
lipid solutions in a round bottom flask. The solvent was rapidly removed using a rotary
evaporator at 65°C (Buchi R-3000, Flawil, Switzerland). The lipid film was then dried for at
least 4 hours in a dessicator under vacuum with ~100g Drierite™ in the base. The lipid film was
then hydrated with the appropriate buffer and vortexed 1 minute to remove all lipids from the
glass walls and suspend them. The lipid solution was then transferred to a test tube and
subjected to five freeze-thaw cycles using liquid N2 (or a dry ice/acetone bath), then room
temperature water, then ~40°C water. This was done to promote vesicle fusion, creating large
multilamellar vesicles, and assisting dye encapsulation (for CF and ANTS/DPX vesicles). A
high-pressure extruder (10 mL water-jacketed Lipex Extruder, Lipex Biomembranes, Vancouver,
CAN) was assembled with two stacked Nucleopore 0.1 µm polycarbonate filters (Whatman,
Florham, NJ), and rinsed once with 10 mL MOPS buffer, then ten times with 1.5 mL MOPS
buffer each. The lipid solution was then extruded ten times at 450 psi, to create homogeneous,
100 nm unilamellar vesicles. CF and ANTS/DPX vesicles were subsequently passed through a
Sephadex® G-25 column to remove unencapsulated dye.
Lipid Concentration Determination
A modified Bartlett assay was used to determine the phosphate concentration of the lipid
solutions and hence, the lipid concentration (28). Briefly, 1.053 mM phosphate standard was
pipetted in 60 µL increments from 0 to 300 µL along with vesicle solution samples in glass test
tubes. All were pipetted in triplicate with the balance as water to keep volumes equal. 700 µL of
17
70% perchloric acid was added and the solutions were heated under reflux at 210°C in an
aluminum heating block for 1 h, to cleave phosphate headgroups from the phospholipids. This
was allowed to cool, then 2.00 mL of 1.0% (w/v) ammonium molybdate/water solution was
added to each tube and vortexed for 1 s. Next, 2.00 mL of 4.0% (w/v) ascorbic acid solution was
added to each tube and vortexed again for 1 s. The tubes were then placed in a 37°C water bath
for 45 minutes, allowing formation of the phosphomolybdate complex. The absorbance was then
read at 580 nm using a spectrophotometer (Spectronic 20D, Thermo Scientific, Waltham, MA).
The unknown lipid concentration was determined using the linear regression of the phosphate
standards.
7-Methoxycoumarin Lipid Probe Synthesis
7MC-POPE (7MC amide linked to the POPE headgroup) was synthesized using the Vaz
and Hallman method (29). Briefly, all organic solvents were dried for 24 h using molecular
sieves (Sigma-Aldrich, St. Louis, MO). Approximately 700 µL stock POPE stored in CHCl3
was dried with rotary evaporation then redissolved in 700 µL dry CHCl3. Next, 10 mg 7MC was
dissolved in dry DMF and added to an aluminum foil-wrapped test tube, followed by 2.3mg of
ground, dry K2CO3. The 700 µL POPE (of ~[60 µM]) solution was then added to the mixture
which was stirred in the dark until the reaction was complete (usually less than 2 h). The
reaction was monitored by SiO2 TLC using 65:25:4 (v/v/v) CH2Cl2/MeOH/water. The 7MC was
visualized by UV lamp, and the lipid by Zinzade’s reagent followed by ashing (30). The product
was purified by either preparatory SiO2 TLC or SiO2 column using 2:1 (v/v) CH2Cl2/MeOH.
7MC-POPE was then dried using rotary evaporation then redissolved in a minimal volume of dry
CHCl3. Concentration of the lipid probe was determined using a spectrophotometer (Cary 1E
18
UV-Vis, Varian, St. Helens, Australia), using the absorption maximum at 348 nm and ε =
2.9×104 M
-1 cm
-1.
Fluorescence Experiments
Kinetic Binding Experiments. Binding kinetics for CE2, MG2, and TP2 were determined
using an SX.18MV-R stopped-flow fluorometer (Applied Photophysics, Leatherhead, United
Kingdom). Binding vesicles were created using various compositions of POPC and POPG, also
including 2 mol% 7MC-POPE. Excitation was set at 280 nm and emission was monitored
through a 385 nm long-pass filter by Edmund Industrial Optics (Barrington, NJ). This emission
wavelength was chosen because the emission maximum of 7MC-POPE at 396 nm, is the same as
7MC. The overlap of the tryptophan emission and 7MC absorption is shown in Figure 6, and a
schematic of the energy transfer is shown in Figure 7. Experiment concentrations were always 1
µM peptide and lipid concentration normally varied between 25 and 200 µM.
19
Figure 6: Absorption and emission spectra of tryptophan and 7MC. Overlap of the emission of tryptophan and the
absorption of 7MC is the necessary condition which allows FRET to occur.
20
Figure 7: Schematic of fluorescence resonance energy transfer from tryptophan to 7-methoxycoumarin. When the
intrinsic tryptophan is greater than the Förster distance from 7MC, no energy transfer can occur (left). As the
peptide binds and interacts with the membrane, energy transfer to the 7MC occurs and can be monitored (right).
21
Analysis of Binding. Binding analysis was performed based on the work of Gregory et al.
(13,25). Briefly, the reversible binding of a peptide-lipid complex can be described by the
equilibrium expression, Eq. 1.
(1)
Here P is peptide, L is lipid, PL is the peptide-lipid complex, kon is the on-rate constant and koff
is the off-rate constant. The peptide-lipid complex formation can be described as a differential
function of time as seen in Eq. 2:
(2)
Here, the bracketed terms represent concentrations of the lipid, peptide and peptide-lipid
complex. Assuming [P] << [L], and that the lipid concentration is constant (lipids are not
consumed in these analyses), the rate law described is pseudo 1st order. The data were fit with
the time-dependent solution to Eq. 2, a one-exponential curve, described as Eq. 3:
(3)
These experiments are conducted in non-equilibrium conditions and hence the apparent rate
constants, kapp, were calculated using Eq. 3. The constants C1 and C2 correspond to the intensity
(amplitude) and fluorescence signal value at time = 0 (y-intercept), respectively. The apparent
rate constant can be expressed as the linear relationship in Eq. 4:
(4)
and the equilibrium dissociation constant, KD, written as:
22
. (5)
Carboxyfluoroscein Efflux Experiments. LUVs were prepared by hydrating mixed
compositions of POPC/POPG lipid films using CF buffer. Fluorescence was monitored over
time using an SX.18MV-R stopped-flow fluorometer (Applied Photophysics, Leatherhead,
United Kingdom). As peptides perturb the vesicle, the self-quenching encapsulated CF is
released into the surrounding buffer, and is diluted, leading to an increase in fluorescence signal.
Excitation was set at 470 nm and emission measured through a 530 nm long-pass filter (Edmund
Industrial Optics, Barrington, NJ). Peptide concentration was always 1 µM and lipid
concentration was varied from 25 to 200 µM. The fractional release was normalized using the
maximum fluorescence levels determined by dissolving the vesicles using 1% Triton X-100,
comparing fluorescence of the solution before and after complete vesicle disruption. Due to
error and variances in technique and instrumentation, ―full release‖ is loosely defined as when a
peptide induces fractional CF release between ~80 and 100%.
ANTS/DPX Assay. The ANTS/DPX assay was performed to discover the mechanism of
peptide action, as described in detail by Ladohkin et al. (26,27). LUVs were prepared by
hydrating mixed compositions of POPC/POPG lipid films using ANTS/DPX hydration buffer.
Fluorescence measurements were recorded in an SLM-Aminco 8100 spectrofluorimeter
upgraded with monochromator stepper and photon counter modules by ISIS. Excitation was set
at 365 nm, with 1 nm slit width, and emission monitored at 515 nm, using an 8 nm slit width.
Vesicle concentration was constant in the experiments, and estimated at 600 µM. Varying
volumes of peptide solution were added depending on the concentration of the solution, but
usually in the range of 1 to 30 µL.
23
Circular Dichroism Experiments
Circular dichroism experiments were performed using a Chirascan (Applied
Photophysics, Leatherhead, United Kingdom). The secondary structure of peptides was
calculated both free in solution and bound to various composition vesicles. Experiments were
performed in a 1 mm quartz cell at 25°C, using 1 nm steps from 190 to 260 nm and corrected for
background signal from buffer and vesicles. The estimated peptide helicities were determined
using the spectral minimum at 222 nm (31):
(6)
where [Θ222] is the molar ellipticity at 222 nm, expressed as deg∙cm2∙dmol
-1 (32), and [Θcoil] and
[Θhelix] are given by Eqs. 7 and 8 (33):
(7)
(8)
The parameter n is the number of amino acids in the peptide sequence and T represents
temperature in °C.
24
RESULTS
Kinetic Binding Experiments
Binding of CE2 and MG2 was measured by fluorescence resonance energy transfer
(FRET) between the tryptophan incorporated in all of these peptide sequences and 7MC-POPE.
Excitation of tryptophan at 280 nm causes fluorescence at 350 nm. The tryptophan of a peptide
within the Förster distance to a 7MC labeled phospholipid incorporated in the lipid membrane,
can transfer this energy to 7MC, consequently fluorescing at 396 nm (see Figures 6 and 7).
Since this energy transfer is a function of distance between the FRET pair and only occurs when
both are about 28Å apart or less, increase in fluorescence at 396 nm is indicative of the binding
event.
The binding data were fit with Eq. 3, and kapp was plotted as a function of lipid
concentration. Linear regression of these plots gave kon (the slope) and koff (the y-intercept) via
Eq. 4, further allowing calculation of KD. Modification of regression fits was sometimes
necessary to account for deviation in certain constants. If the y-intercept is especially close to
the origin, significant error in the experimental value for koff may exist. If this is the case,
dissociation experiments should be performed to establish an accurate value for the off-rate
constant.
At high peptide to lipid ratios, the kinetics of binding deviate from linearity. The
numerous peptides per vesicle tend to disrupt the vesicle catastrophically, causing micellization
or destroying the integrity of the vesicle and the lipid bilayer. Since peptide concentration is
held constant, this occurs near the lower lipid concentration range in these experiments, around
25 µM and below. At such low lipid concentrations, the peptide:vesicle ratio can approach
5000:1 and the results can be seen in Figure 8.
25
Figure 8: Lipid concentration effects on binding. This plot of kapp as a function of lipid concentration for CE2
shows that as the lipid goes below ~30 µM, the curve deviates from linearity. The interactions between the peptides
and vesicles may lead to micellization or other catastrophic events for the vesicles, affecting the binding constants at
low lipid concentration. The solid line is the linear fit for all points, the dashed line is fit to the region 30 to 200 µM,
and the dotted line is fit from 5 to 30 µM.
26
CE2 Binding
CE2 binding to pure POPC vesicles was marginal, thus binding had to be performed on
various compositions of vesicles in order to determine binding constants for pure POPC. Vesicle
compositions of 50:50:2 up to 90:10:2 POPC/POPG/7MC-POPE were used. A representative
example of the data collected in these experiments is shown in Figure 9. Various graphs of KD,
kon, and koff as a function of vesicle POPC content were used to extrapolate values for pure POPC
vesicles, and are seen in Figures 10 and 11. The binding kinetic constants and dissociation
constants are compared for cecropin A and CE2 in Table 2.
27
Figure 9: Kinetics of CE2 binding to vesicles of POPC/POPG 50:50 (A and B), 70:30 (C and D), 80:20 (E and F),
and 90:10 (G and H). On the left, the curves represent ~20 averaged experimental binding kinetics traces at 25 µM
lipid and 1 µM CE2, and the curve is the single-exponential fit to the data (Eq. 3). To the right of each graph is the
apparent rate constant plotted as a function of lipid concentration, yielding koff from the y-intercept and kon from the
slope (Eq. 4)
28
Figure 10: (A) CE2 on-rates as a function of POPC content in mixed POPC/POPG vesicles. The binding
experiments were performed with 1µM CE2 and various lipid concentrations from 25 to 200 µM. Increasing the
neutral character of vesicles causes marked decrease in the affinity of the peptide for the vesicle. When the
composition reaches a certain point, around 80 to 90%POPC, the on-rate is greatly affected and deviates from the
other data. (B) CE2 off-rates as a function of POPC content in mixed POPC/POPG vesicles. The off-rate at 100%
POPC can be calculated based on a fit of the data through 80% POPC. Above this point, binding is weak, and the
off-rate is artificially deflated, seen at 90% POPC.
29
Figure 11: (A) CE2 equilibrium dissociation constant as a function of POPC content in POPC/POPG mixed
vesicles. The exponential fit is weighted based on the variance of the KD values and shows the extrapolation at
100% POPC. (B) The natural logarithm of the equilibrium dissociation constant as a function of POPC content in
POPC/POPG mixed vesicles. Again, the regression can be used to extrapolate the 100% POPC value for KD. The
deviation of koff at high POPC percentage leads to deflated KD values, seen in the 90% value.
30
Table 2: Cecropin A and CE2 On- and Off-Rate Constants and Equilibrium Dissociation Constants
Vesicle composition Peptide Binding kinetics Dissociation constant*
POPC/POPG kon (M-1
s-1
) koff (s-1
) KD (µM)
50:50 Cecropin A
CE2 (5.2 ± 0.2) 10
5
(6.4 ± 0.1) 105
7 ± 2
15 ± 2
13 ± 4
23 ± 3
70:30 Cecropin A
CE2 (4.3 ± 0.4) 10
5
(2.3 ± 0.2) 105
46 ± 4
86 ± 3
110 ± 14
377 ± 35
80:20 Cecropin A
CE2 (3.9 ± 4.0) 10
5
(1.7 ± 0.7) 105
170 ± 40
100 ± 10
440 ± 460
700 ± 300
90:10
100:0‡
Cecropin A
CE2
Cecropin A
CE2
ND†
(8 ± 2) 104
2 105
9.4 104
ND
81 ± 3
300
370
—
1100 ± 280
1000
5000
*KD is calculated solely from binding kinetics and expressed as a function of lipid concentration, not vesicle
concentration. †These values were not determinable due to weak signal. ‡Values for 100% POPC vesicles were
extrapolated from the best-fit curves for cecropin A and CE2.
31
MG2 Binding
MG2 binding to pure POPC vesicles was also marginal, but better than the parent
peptide, thus binding at 100% POPC could be measured, and values obtained for kon, koff, and KD
for pure POPC vesicles were experimental rather than extrapolated. Vesicle compositions of
50:50:2 up to 90:10:2 POPC/POPG/7MC-POPE were used in addition to 100:2 POPC/7MC-
POPE. The collected fluorescence data from these experiments are shown in Figure 12. Various
graphs of KD, kon, and koff as a function of vesicle POPC content show the data fit and values for
100% POPC vesicles, shown in Figures 13 and 14. The kinetic constants for binding and
dissociation are compared for magainin 2 and MG2 in Table 3.
32
Figure 12: MG2 binding data. Curves in (A), (C), (E), and (G) represent raw fluorescence data from binding at 25
µM lipid and 1 µM peptide concentration. Vesicle composition is POPC/POPG/7MC-POPE 50:50:2 in (A), 70:30:2
in (C), 80:20:2 in (E), and 100:2 (POPC:7MC-POPE) in (G). All curves are the average of ~20 curves and the gray
line is the one-exponential fit. To the right of each graph is the corresponding linear regression of the apparent rate
constant to the lipid concentration, yielding kon and koff for each vesicle composition.
33
Figure 13: (A) MG2 on-rate (A) and off-rate (B) constants as a function of POPC content in mixed POPC/POPG
vesicles. The binding experiments were performed with 1µM CE2 and various lipid concentrations from 25 to 200
µM. Increasing the neutral character of vesicles causes marked decrease in the affinity of the peptide for the vesicle.
Linear regression of the data is shown in each graph.
34
Figure 14: MG2 equilibrium dissociation constant as a function of POPC content in POPC/POPG mixed vesicles.
MG2 bound sufficiently well that KD values were experimentally determined for 100% POPC vesicles. The top
graph shows the data plotted using a linear scale. The bottom graph shows the natural log of the data and the linear
correlation in this form. The lines represent the best data fit in each graph.
35
Table 3: Magainin 2 and MG2 On- and Off-Rate Constants and Equilibrium Dissociation Constants
Vesicle composition Peptide Binding kinetics Dissociation constant*
POPC/POPG kon (M-1
s-1
) koff (s-1
) KD (µM)
50:50 Magainin 2
MG2 (9.1 ± 0.4) 10
5
(6.5 ± 0.6) 105
20 ± 9
26 ± 3
22 ± 10
40 ± 6
70:30 Magainin 2
MG2 (8.5 ± 0.7) 10
5
(5.6 ± 0.8) 105
77 ± 14
27 ± 3
91 ± 18
49 ± 9
80:20 Magainin 2
MG2 (7.8 ± 0.7) 10
5
(3.6 ± 0.6) 105
160 ± 15
37 ± 1
205 ± 27
103 ± 17
90:10
100:0‡
Magainin 2
MG2
Magainin 2
MG2
(5.5 ± 0.9) 105
(1.9 ± 0.5) 105
9.7 104
(0.2 ± 0.2) 104
350 ± 20
25 ± 6
600
23 ± 2
640 ± 110
126 ± 45
6000
2000 ± 1200
*KD is calculated solely from binding kinetics and expressed as a function of lipid concentration, not vesicle
concentration. ‡Values for 100% POPC vesicles were extrapolated from the best-fit curves for magainin 2.
36
Carboxyfluorescein Efflux Experiments
Carboxyfluorescein dye (CF) was encapsulated in large unilamellar vesicles at self
quenching concentrations. Mixing peptide with the CF loaded vesicles permeabilized the
vesicles and caused the CF to leak into the surrounding buffer. This dilution of the CF relieved
the self-quenching and the increase in CF fluorescence was measured over time. Peptide
concentration in all experiments was held constant at 1 µM and lipid concentration was varied
from 25 to 200 µM. Variances in technique and instrumentation require definition of ―full
release,‖ and this was established as causing fractional release in the range of ~0.8 to 1.
Early experiments indicated that the fractional release of CF was notably lower than
expected for CE2 compared to cecropin A (13). The initial interaction of CE2 and vesicles was
not being captured in the long runs due to excessive signal averaging. Thus, split timebases were
used in order to capture data more frequently in the initial mixing period, and at longer intervals
afterwards. The result was fractional release at the expected value, or full release. We see in
Figure 15 that CE2 releases all dye from the vesicles; however, at high lipid concentrations, the
efflux takes much longer than native cecropin A. For 50:50 POPC/POPG vesicles, at 25 and 50
µM, the rate of release for CE2 and cecropin A are about the same. At 100 µM, CE2 takes about
600 s to release 80% of the dye where cecropin A only took 400 s to achieve this same release
level. At 200 µM, CE2 takes about 2500 s to invoke 80% release where cecropin A takes about
650 s. But, these differences in efflux rates are minimal when considering potential error
accumulation in these experiments.
37
Figure 15: 1 µM CE2 dye efflux experiments at various concentrations of 50:50 POPC/POPG vesicles. The curves
each represent one experiment with 25, 50, 100, and 200 µM lipid concentrations. The fastest curve is 25 µM and
the slowest curve is 200 µM. Notable is the extremely rapid initial efflux, as well as the induced complete release
(top). There is significant fractional CF release in the short time period immediately following mixing of peptide
and vesicles, and this sharp increase seems to be concentration independent (bottom).
38
MG2 induced complete release of vesicles at 50:50 and 70:30 POPC/POPG but at a much
faster rate than magainin 2. The efflux curves for MG2 can be seen in Figure 16. Using 50:50
POPC/POPG vesicles, MG2 produced 80% release for 25 µM lipid in about 1 second where it
took Magainin 2 about 250 s. At 50 µM lipid for the same composition, it took MG2 about 3
seconds to elicit 80% release where Magainin 2 took 750 s. Using 70:30 POPC/POPG vesicles,
at 25 µM lipid, MG2 produced 80% release in about 10 s and magainin 2 took about 2500 s. At
50 µM lipid, MG2 elicited 80% release in about 20 s, and magainin 2 took about 4000 s.
Changing the lipid composition to 100% POPC caused the efflux rate to diminish greatly, as seen
in Figure 17.
39
Figure 16: 1 µM MG2 dye efflux experiments at various concentrations of 50:50 and 70:30 POPC/POPG vesicles.
The top graph is for 50:50 POPC/POPG and the bottom graph is for 70:30 POPC/POPG. The curves each represent
one experiment with 25, 50, 100, and 200 µM lipid concentrations where the fastest curve is 25 µM and the slowest
curve is 200 µM.
40
Figure 17: 1 µM MG2 dye efflux experiment with 50 µM 100% POPC vesicles. Even with a five hour timescale,
efflux was not complete, showing the significant effect of incorporating negative phospholipids in these vesicles for
MG2.
41
ANTS/DPX Assay
The mechanism of dye release by CE2 was measured using the ANTS/DPX assay.
Vesicles were prepared by incorporating the fluorophore/quencher pair ANTS and DPX. If
release is all-or-none, then the fluorescence quenching inside the vesicles is independent of the
peptide concentration. The plot of quenching inside the vesicles (Qin) as a function of
fluorescence outside the vesicles (fout) for all-or-none peptides is a horizontal line. CE2 causes
graded release where cecropin A causes all-or-none release. MG2 also causes graded release and
magainin-2 causes all-or-none release.
The mechanism of dye release by CE2 and MG2 was determined using the ANTS/DPX
assay and this data is represented in Figures 18 and 19 respectively. Vesicles were prepared by
encapsulating the fluorophore/quencher pair ANTS and DPX. If the mechanism of release is all-
or-none, all the contents of some vesicles is released, and the fluorescence quenching inside the
vesicles is independent of the peptide concentration. Graded release causes the vesicles to leak
part of their contents; however, usually the release of ANTS and DPX is unequal, and the
fluorescence inside the vesicles increases. The plot of quenching inside the vesicles (Qin) as a
function of fluorescence outside the vesicles (fout) for all-or-none peptides is a horizontal line and
for graded peptides the plot is a rising curve. The fit equations used for these experiments are
those established by Ladokhin et al., and shown in Eqs. 9a and 9b (26,27).
(9a)
(9b)
42
and are the fluorescence intensities inside the vesicles without quencher.
represents the initial concentration of DPX which was 8mM, is the portion of ANTS
outside the vesicles, is the dynamic quenching constant, 50 M-1
(26,27), is the static
quenching constant (a fit value from Eq. 9b), and α (also fit from Eq. 9b) is the ratio of DPX
released to ANTS released. Since DPX is positively charged, the incorporation of negatively
charged POPG in the vesicles is likely influencing the shape of the curve relative to previous
experiments. Selective release of DPX is apparent, with an α value of 3 for the MG2 experiment
where 70:30 POPC/POPG vesicles were used, and an α value of 9 for the CE2 experiments
where 50:50 POPC/POPG vesicles were used.
43
Figure 18: ANTS/DPX assay from three experiments for CE2, showing graded mechanism for ~600 µM 50:50
POPC/POPG lipid vesicles with varying concentrations of CE2. If the mechanism were all-or-none, the data would
have generally followed the dashed line. The sigmoidal shape of the curve, governed by the fit parameter α in Eq.
9b, indicates high propensity for selective release of DPX, probably due to the negative character of the membrane.
The fit parameters for Eq. 9b were α = 8.6, and Ka = 188.
44
Figure 19: ANTS/DPX assay for MG2 using ~600 µM 70:30 POPC/POPG vesicles. Again, the sigmoidal shape is
probably attributed to the large amount of POPG in the vesicles. The fit parameters for Eq. 9b were α = 3.1, and Ka
= 192.
45
Circular Dichroism Experiments
The secondary structure of the peptides was determined by circular dichroism, CD, for
mixed compositions of POPC/POPG vesicles. A buffer containing PO43-
, pH 7.5 was used to
dilute the vesicles. Mixed with a high concentration of 50:50 POPC/POPG vesicles, CE2 was
69% and MG2 was 42% helical. Helicities with 100% POPC vesicles at high lipid concentration
were 35% for CE2 and 21% for MG2, but these values are not corrected for the high portion of
unbound peptide. The CD data for CE2 can be seen in Figure 20, and MG2 CD data can be seen
in Figure 21.
46
Figure 20: CD spectra for CE2 using 50:50 POPC/POPG (A) and 100% POPC (B) vesicles.
47
Figure 21: CD spectra for MG2 using 50:50 POPC/POPG (A) and 100% POPC (B) vesicles.
48
Thermodynamics of Peptide-Lipid Interactions
Thermodynamic values for peptide-LUV interactions were calculated as in previous work
in our lab. The experimental values for the Gibbs free energy of binding, ΔGbind(exp), were
calculated using the KD values of CE2 and MG2 in Eq. 10:
ΔGbind(exp) = RT ln KD – 2.4 kcal/mol (10)
Here R is the gas constant, T is the absolute temperature in Kelvin, and the term 2.4 kcal/mol is
the cratic correction for mixing (10, 34). The Gibbs energy of binding to the membrane-water
interface, ΔGif, is calculated using the Wimley-White interfacial scale (35, 36). The binding
process couples the association of the unstructured peptide with the lipid bilayer and the folding
of this peptide into an α-helix (34). Therefore, ΔGif is the sum of two terms: binding in an
unstructured state and folding on the membrane. ΔGif was calculated based on the determined
helicities of the peptides. Estimation of the Gibbs energy of transfer of the peptide from the
membrane interface to the bilayer interior was done based on the whole-residue octanol transfer
scale (37). Admittedly, the bilayer interior is not ideally represented by octanol, but this
estimation is appropriate in this application (38). The complete thermodynamic cycle is given as
the sum of the terms in Eq. 11:
ΔGins = ΔGf + ΔGoct – ΔGif. (11)
Since the value for ΔGf is very small compared to the other terms, we get the relationship in Eq.
12:
ΔGins ≈ ΔGoct – ΔGif = ΔGoct-if. (12)
49
The experimental and theoretical Gibbs energies of binding are mostly the same. Discrepancies
can arise when the peptide may form salt bridges, which increases the stability of the folded
peptide (10). The thermodynamic calculations were performed with Membrane Protein Explorer
(39) and can be seen in Table 4.
50
Table 4: Thermodynamic Parameters for Peptide Binding and Insertion into POPC Bilayers at Room Temperature
Peptide KD*
ΔGbind(exp) (kcal/mol)
ΔGif(calcd) (kcal/mol)
% helix ΔGoct (kcal/mol)
ΔGoct-if
(kcal/mol)
cecropin A 1 mM -6.4 -2.7 70 31.1 33.8
CE2 5 mM -5.5 -4.3 69 31.2 35.5
magainin 2 6 mM -5.4 -6.0 83 20.3 26.3
MG2 2 mM -6.1 -2.8 42 19.3 22.1
*KD values were calculated with only binding kinetics on- and off-rate constants.
51
DISCUSSION
Mechanism of Vesicle Perturbation
One of the most intriguing discoveries in this research was the difference in the
mechanisms by which the mutant peptides disrupt vesicles compared with the parent peptide.
Cecropin A and magainin 2 disrupt vesicles by means of an all-or-none mechanism (13,14,25).
However, from the ANTS/DPX assay, both CE2 and MG2 undergo a graded mechanism. The
exact reason remains elusive, however, there are some hypotheses. The calculated Gibbs free
energy of insertion, ΔGins, for CE2 is 36 kcal/mol using a peptide helicity of 69% upon binding.
Being this high, one would expect an all-or-none mechanism as with the parent peptide.
However, CE2 is unique in that it contains two, separate helical regions. The helix with the
higher hydrophobic moment may be anchoring itself on the surface of the buffer-vesicle
interface and the other helix may be ―dipping‖ into the bilayer, disturbing the integrity of the
vesicle and allowing contents to steadily leak out in graded fashion. One stipulation in
developing an appropriate antimicrobial peptide is the strength of binding. If binding is too
strong, it may hinder the ability of a peptide to translocate or form the appropriate surface defects
in the membrane to cause leakage.
The free energy of insertion for MG2 is 22 kcal/mol, based on a vesicle-bound helicity of
42%. This is near the cutoff value 20 kcal/mol, but is sufficiently close to deem this a ―gray
zone‖ (10). It is difficult to pinpoint the exact reason for the difference in the mechanism.
Magainin 2, with a ΔGins of 26 kcal/mol with 100% POPC vesicles, has a helicity of 83%.
Experimentally, MG2 had a 20% helicity with pure POPC vesicles, however, this is an
uncorrected value not accounting for unbound peptide. Using the estimate of 42% helicity, the
52
ΔGins of 22 kcal/mol is reasonably close to the 20 kcal/mol border and graded release is not
unreasonable.
Kinetics of Binding
The binding observed with CE2 is in line with our estimations. The binding kinetics
were mostly less than a factor of two different. The difference in dissociation constants is one of
the most notable differences, extrapolated to 1mM for cecropin A and 5mM for CE2, but all
dissociation constants were larger for CE2, indicating weaker binding compared to cecropin A.
The dipping event mentioned above for CE2 may be responsible for the deviation of some
kinetic binding traces from one exponential fits. However, without an estimate of the rate of
insertion, or a method to verify if it does indeed happen, we cannot conclude that this is the only
concurrent process affecting the data.
Binding for MG2 is tighter than for magainin 2, with dissociation constants of 2 mM and
6 mM respectively. The difference is about two-fold for most lipid compositions. One reason
MG2 may be binding better is because the off-rate constant is independent of the negative lipid
character of the vesicles (Figure 13). Another consideration is the differences in helicity of the
native and modified peptides. Magainin 2 has a helicity of about 56% when bound to 50:50
POPC/POPG membranes where MG2 only has a helicity of 42% to the same vesicle
composition. This affects the values obtained for the Gibbs free energy of insertion for the
peptides.
Thermodynamics of Binding
Peptide mechanism does not appear to be limited by thermodynamic constraints only. The Gibbs
free energy of insertion of 36 kcal/mol for CE2 suggests that it should operate with an all-or-
53
none mechanism. Although the complete release in efflux experiments support this, the data
from the ANTS/DPX assay imply that CE2 is a graded release peptide.
The effect of helicity on the calculated free energies is also important going forward in
novel peptide development. The markedly decreased helicity of MG2 compared with magainin 2
shifted the Gibbs energy of insertion into the ―gray zone,‖ making it difficult to pinpoint the
differences in the peptides.
Peptide Efflux
Cecropin A and CE2 both induce complete release of vesicle contents, independent of the
difference in mechanisms. Although the rate of release differs slightly for higher concentrations
of lipid in CE2, they still match reasonably well to the rates of release for cecropin A.
Both magainin 2 and MG2 induce complete release, but MG2 efflux is about 250 times
faster. It is interesting that a graded release by MG2 also produces complete release (one would
expect efflux to cease once the mass imbalance dissipated).
Final Thoughts
Although binding is similar for both peptides, the lowered ΔGins for MG2 is probably
responsible for the change in mechanism compared with magainin 2. However, this does not
explain the complete release seen in efflux experiments. Also, it is intriguing that MG2releases
vesicle contents so much faster than magainin 2, even though it has a much lower helicity.
For CE2 the ΔGins is well outside the region where one would expect a graded release
mechanism, and again, the complete release observed is counterintuitive. Another interesting
point is the CE2 on-rate constant dependence on POPC content in vesicles. One might expect an
54
exponential decrease in the on-rate as POPG content is decreased in the vesicles. It appears there
is a sharp transition where the kon decreases between 70 and 80% POPC content.
Studies of peptide translocation and pore states may help confirm the mechanisms
determined for CE2 and MG2. Design and analysis of helical properties of the peptides and the
effects on peptide effectiveness may also warrant investigation.
55
FUTURE WORK
In order to verify whether peptides which undergo all-or-none mechanisms, it would be
necessary to perform experiments which test for translocation of the peptides. If an all-or-none
peptide were to translocate, it could produce graded results and may cease efflux at a certain
level by relieving the mass imbalance across the membrane. Further, testing lipid flip-flop rates
and their effects on the translocation or lack thereof would also support the determined
mechanisms of MG2 and CE2.
Another means to verify the results of the thermodynamic values calculated in this
experiment would be through differential scanning calorimetry and isothermal titration
calorimetry. These methods could support or refute the results of this research based on the
obtained energy values for the binding of these peptides.
Analysis of the pore state lifetimes would also help verify the mechanism for these
peptides. If the pores are long-lived, it would support the all-or-none mechanism and if they are
short-lived, a graded mechanism would be indicated. This would help to confirm that the
mechanism of release for CE2 is indeed graded, and not the results of another, unknown process.
56
ABBREVIATIONS
POPC, 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; POPG, 1-palmitoyl-2-oleoyl-sn-
glycero-3-phospho-(1’-rac-glycerol); POPE, 1-palmitoyl-2-oleoyl-sn-glycero-3-
phosphoethanolamine; 7MC, 7-methoxycoumarin-3-carboxylic acid; ANTS, 8-
aminonaphthalene-1,3,6-trisulfonic acid; DPX, p-xylene-bis-pyridinium bromide; LUV, large
unilamellar vesicle; FRET, fluorescence resonance energy transfer; CD, circular dichroism;
MPEx, Membrane Protein Explorer; ΔGif, Gibbs energy of peptide binding to membrane-water
interface as a helix; ΔGins, Gibbs energy of insertion from interface into membrane; ΔGbind,
experimentally determined Gibbs energy of binding; ΔGf, Gibbs energy of folding to an α-helix
in water; ΔGoct, Gibbs energy of peptide transfer from water to octanol; ΔGoct-if = ΔGoct - ΔGif;
kon, on-rate constant; koff, off-rate constant; KD, equilibrium dissociation constant.
57
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Purification and properties of three inducible bactericidal proteins from hemolymph of
immunized pupae of Hyalophora cecropia. Eur. J. Biochem. 106, 7-16.
2. Harder, J., Gläser, R., and Schröder, J. (2007) Human antimicrobial proteins – Effectors of
innate immunity. J. Endotoxin Research 13, 317-339.
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