Biomedical subjects

Alan J Waring

Publications and source records attributed to Alan J Waring.

At least 19 recordsLinked to original sources

PubMed2006

Modular determinants of antimicrobial activity in platelet factor-4 family kinocidins.

Mammalian platelets contain an array of antimicrobial peptides, termed platelet microbicidal proteins (PMPs). Human and rabbit PMPs include known chemokines, such as platelet factor-4 (hPF-4); PMP-1 is the rabbit orthologue of hPF-4. Chemokines that also exert direct antimicrobial activity have been termed kinocidins. A consensus peptide domain library representing mammalian PF-4 family members was analyzed to define structural domains contributing to antimicrobial activity against a panel of human pathogens. Secondary conformations were assessed by circular dichroism spectrometry, and molecular modeling was employed to investigate structural correlates of antimicrobial efficacy. Antimicrobial activity against isogenic peptide-susceptible or -resistant Staphylococcus aureus, Salmonella typhimurium, and Candida albicans strain pairs mapped to the C-terminal hemimer (38-74) and modular domains thereof (49-63 and 60-74). Increasing electrostatic charge and steric bulk were general correlates of efficacy. Structural data corroborated spatial distribution of charge, steric bulk and putative secondary structure with organism-specific efficacy. Microbicidal efficacies of the cPMP antimicrobial hemimer and C-terminal peptide (60-74) were retained in a complex human-blood biomatrix assay. Collectively, these results suggest that modular determinants arising from structural components acting independently and cooperatively govern the antimicrobial functions of PF-4 family kinocidins against specific target pathogens.

Amino Acid Sequence↗

PubMed2006

Structural correlates of antimicrobial efficacy in IL-8 and related human kinocidins.

Chemokines are small (8-12 kDa) effector proteins that potentiate leukocyte chemonavigation. Beyond this role, certain chemokines have direct antimicrobial activity against human pathogenic organisms; such molecules are termed kinocidins. The current investigation was designed to explore the structure-activity basis for direct microbicidal activity of kinocidins. Amino acid sequence and 3-dimensional analyses demonstrated these molecules to contain iterations of the conserved gamma-core motif found in broad classes of classical antimicrobial peptides. Representative CXC, CC and C cysteine-motif-group kinocidins were tested for antimicrobial activity versus human pathogenic bacteria and fungi. Results demonstrate that these molecules exert direct antimicrobial activity in vitro, including antibacterial activity of native IL-8 and MCP-1, and microbicidal activity of native IL-8. To define molecular determinants governing its antimicrobial activities, the IL-8 gamma-core (IL-8gamma) and alpha-helical (IL-8alpha) motifs were compared to native IL-8 for antimicrobial efficacy in vitro. Microbicidal activity recapitulating that of native IL-8 localized to the autonomous IL-8alpha motif in vitro, and demonstrated durable microbicidal activity in human blood and blood matrices ex vivo. These results offer new insights into the modular architecture, context-related deployment and function, and evolution of host defense molecules containing gamma-core motifs and microbicidal helices associated with antimicrobial activity.

Amino Acid Motifs↗

PubMed2006

Membrane-bound conformation and topology of the antimicrobial peptide tachyplesin I by solid-state NMR.

The conformation and membrane topology of the disulfide-stabilized antimicrobial peptide tachyplesin I (TP) in lipid bilayers are determined by solid-state NMR spectroscopy. The backbone (phi and psi) torsion angles of Val(6) are found to be -133 degrees and 142 degrees , respectively, and the Val(6) CO-Phe(8) H(N) distance is 4.6 A. These constrain the middle of the N-terminal strand to a relatively ideal antiparallel beta-sheet conformation. In contrast, the phi angle of Gly(10) is +/-85 degrees , consistent with a beta-turn conformation. Thus, TP adopts a beta-hairpin conformation with straight strands, similar to its structure in aqueous solution but different from a recently reported structure in DPC micelles where bending of the two beta-strands was observed. The Val(6) and Gly(10) CO groups are both 6.8 A from the lipid (31)P, while the Val(6) side chain is in (1)H spin diffusion contact with the lipid acyl chains. These results suggest that TP is immersed in the glycerol backbone region of the membrane and is oriented roughly parallel to the plane of the membrane. This depth of insertion and orientation differs from those of the analogous beta-sheet antimicrobial peptide protegrin-1 and suggest the importance of structural amphiphilicity in determining the location and orientation of membrane peptides in lipid bilayers.

Anti-Infective Agents↗

PubMed2006

Trehalose-protected lipid membranes for determining membrane protein structure and insertion.

Trehalose preserves lipid bilayers during dehydration and rehydration by replacing water to form hydrogen bonds between its own OH groups and lipid headgroups. We compare the lipid conformation and dynamics between trehalose-protected lyophilized membranes and hydrated membranes, to assess the suitability of the trehalose-containing membrane as a matrix for membrane protein structure determination. (31)P spectra indicate that the lipid headgroup of trehalose-protected dry POPC membrane (TRE-POPC) have an effective phase transition temperature that is approximately 50K higher than that of the hydrated POPC membrane. In contrast, the acyl chains have similar transition temperatures in the two membranes. Intramolecular lipid (13)C'-(31)P distances are the same in TRE-POPC and crystalline POPC, indicating that the lipid headgroup and glycerol backbone conformation is unaffected by trehalose incorporation. Intermolecular (13)C-(31)P distances between a membrane peptide and the lipid headgroups are 10% longer in the hydrated membrane at 226 K than in the trehalose-protected dry membrane at 253 K. This is attributed to residual motions in the hydrated membrane, manifested by the reduced (31)P chemical shift anisotropy, even at the low temperature of 226 K. Thus, trehalose lyoprotection facilitates the study of membrane protein structure by allowing experiments to be conducted at higher temperatures than possible with the hydrated membranes.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2006

Mechanism of supported membrane disruption by antimicrobial peptide protegrin-1.

While pore formation has been suggested as an important step in the membrane disruption process induced by antimicrobial peptides, membrane pore formation has never been directly visualized. We report on the dynamics of membrane disruption by antimicrobial peptide protegrin-1 (PG-1) on dimyristoyl-sn-glycero-phosphocholine-supported bilayer patches obtained via atomic force microscopy. The action of PG-1 is found to be concentration-dependent. At low PG-1 concentrations (1 < [PG-1] < 4 microg/mL), the peptide destabilizes the edge of the membrane to form fingerlike structures. At higher concentrations, PG-1 induces the formation of a sievelike nanoporous structure in the membrane. The highest degree of disruption is attained at concentrations >or=20 microg/mL, at which PG-1 disrupts the entire membrane, transforming it into stripelike structures with a well-defined and uniform stripe width. This first direct visualization of these membrane structural transformations helps elucidate the PG-1-induced membrane disruption mechanism.

Antimicrobial Cationic Peptides↗

PubMed2006

Membrane-dependent oligomeric structure and pore formation of a beta-hairpin antimicrobial peptide in lipid bilayers from solid-state NMR.

We used solid-state NMR spectroscopy to investigate the oligomeric structure and insertion of protegrin-1 (PG-1), a beta-hairpin antimicrobial peptide, in lipid bilayers that mimic either the bacterial inner membrane [palmitoyloleoylphosphatidyl ethanolamine and palmitoyloleoylphosphatidylglycerol (POPE/POPG) bilayers] or the red blood cell membrane [neutral palmitoyloleoylphosphatidylcholine (POPC)/cholesterol bilayers]. (1)H spin diffusion from lipids to the peptide indicates that PG-1 contacts both the lipid acyl chains and the headgroups in the anionic membrane but resides far from the lipid chains in the POPC/cholesterol bilayer. (19)F spin diffusion data indicates that 75% of the beta-hairpins have homodimerized N strands and C strands in the anionic membrane. The resulting (NCCN)(n) multimer suggests a membrane-inserted beta-barrel enclosing a water pore. The lipids surrounding the beta-barrel have high orientational disorder and chain upturns, thus they may act as fillers for the pore. These results revise several features of the toroidal pore model, first proposed for magainin and subsequently applied to PG-1. In the POPC/cholesterol membrane, the N and C strands of PG-1 cluster into tetramers, suggesting the formation of beta-sheets on the membrane surface. Thus, the membrane composition plays a decisive role in defining the assembly and insertion of PG-1. The different oligomeric structures of PG-1 help to explain its greater toxicity for bacteria than for eukaryotic cells.

Anions↗

PubMed2006

Insertion selectivity of antimicrobial peptide protegrin-1 into lipid monolayers: effect of head group electrostatics and tail group packing.

The ability to selectively target the harmful microbial membrane over that of the host cell is one of the most important characteristics of the antimicrobial peptides (AMPs). This selectivity strongly depends on the chemical and structural properties of the lipids that make up the cell membrane. A systematic study of the initial membrane selectivity of protegrin-1 (PG-1), a beta-sheet AMP, was performed using Langmuir monolayers. Constant pressure insertion assay was used to quantify the amount of PG-1 insertion and fluorescence microscopy was employed to observe the effect of PG-1 on lipid ordering. Charge and packing properties of the monolayer were altered by using lipids with different head groups, substituting saturated with unsaturated lipid tail group(s) and incorporating spacer molecules. PG-1 inserted most readily into anionic films composed of phosphatidylglycerol (PG) and lipid A, consistent with its high selectivity for microbial membranes. It also discriminated between zwitteranionic phospholipids, inserting more readily into phosphatidylcholine (PC) monolayers than those composed of phosphatidylethanolamine, potentially explaining why PG-1 is hemolytic for PC-rich human erythrocytes and not for the PE-rich erythrocytes of ruminants. Increased packing density of the monolayer by increased surface pressure, increased tail group saturation or incorporation of dihydrocholesterol diminishes the insertion of PG-1. Fluorescence microscopy shows that lipid packing is disordered upon PG-1 insertion. However, the presence of PG-1 can still affect lipid morphology even with no observed PG-1 insertion. These results show the important role that lipid composition of the cell membrane plays in the activity of AMPs.

Anti-Infective Agents↗

PubMed2006

Retrocyclins kill bacilli and germinating spores of Bacillus anthracis and inactivate anthrax lethal toxin.

Theta-defensins are cyclic octadecapeptides encoded by the modified alpha-defensin genes of certain nonhuman primates. The recent demonstration that human alpha-defensins could prevent deleterious effects of anthrax lethal toxin in vitro and in vivo led us to examine the effects of theta-defensins on Bacillus anthracis (Sterne). We tested rhesus theta-defensins 1-3, retrocyclins 1-3, and several analogues of RC-1. Low concentrations of theta-defensins not only killed vegetative cells of B. anthracis (Sterne) and rendered their germinating spores nonviable, they also inactivated the enzymatic activity of anthrax lethal factor and protected murine RAW-264.7 cells from lethal toxin, a mixture of lethal factor and protective antigen. Structure-function studies indicated that the cyclic backbone, intramolecular tri-disulfide ladder, and arginine residues of theta-defensins contributed substantially to these protective effects. Surface plasmon resonance studies showed that retrocyclins bound the lethal factor rapidly and with high affinity. Retrocyclin-mediated inhibition of the enzymatic activity of lethal factor increased substantially if the enzyme and peptide were preincubated before substrate was added. The temporal discrepancy between the rapidity of binding and the slowly progressive extent of lethal factor inhibition suggest that post-binding events, perhaps in situ oligomerization, contribute to the antitoxic properties of retrocyclins. Overall, these findings suggest that theta-defensins provide molecular templates that could be used to create novel agents effective against B. anthracis and its toxins.

Amino Acid Sequence↗

PubMed2006

HIV-1 adapts to a retrocyclin with cationic amino acid substitutions that reduce fusion efficiency of gp41.

Retrocyclin (RC)-101 is a cationic theta-defensin that inhibits HIV-1 entry. Passaging HIV-1(BAL) under selective pressure by this cyclic minidefensin resulted in only a 5- to 10-fold decrease in viral susceptibility to RC-101. Emergent viral isolates had three amino acid substitutions in their envelope glycoprotein. One was in a CD4-binding region of gp120, and the others were in the heptad repeat (HR) domains of gp41 (HR1 and HR2). Each mutation replaced an electroneutral or electronegative residue with one that was positively charged. These mutations were evaluated either alone or in combination in a single-round viral entry assay. Although the mutation in gp120 did not affect viral entry, the mutation in HR1 of gp41 conferred relative resistance to RC-101. Interestingly, the envelope with the HR2 mutation was less efficient and became codependent on the presence of RC-101 for entry. The adaptive response of HIV-1 to this cationic host defense peptide resembles the responses of bacteria that modulate their surface or membrane charge to evade analogous host defense peptides. These findings also suggest that interactions between theta-defensins and gp41 may contribute to the ability of these cyclic minidefensins to prevent HIV-1 entry into target cells.

Adaptation, Biological↗

PubMed2006

Theta-defensins prevent HIV-1 Env-mediated fusion by binding gp41 and blocking 6-helix bundle formation.

Retrocyclin-1, a -defensin, protects target cells from human immunodeficiency virus, type 1 (HIV-1) by preventing viral entry. To delineate its mechanism, we conducted fusion assays between susceptible target cells and effector cells that expressed HIV-1 Env. Retrocyclin-1 (4 microm) completely blocked fusion mediated by HIV-1 Envs that used CXCR4 or CCR5 but had little effect on cell fusion mediated by HIV-2 and simian immunodeficiency virus Envs. Retrocyclin-1 inhibited HIV-1 Env-mediated fusion without impairing the lateral mobility of CD4, and it inhibited the fusion of CD4-deficient cells with cells bearing CD4-independent HIV-1 Env. Thus, it could act without cross-linking membrane proteins or inhibiting gp120-CD4 interactions. Retrocyclin-1 acted late in the HIV-1 Env fusion cascade but prior to 6-helix bundle formation. Surface plasmon resonance experiments revealed that retrocyclin bound the ectodomain of gp41 with high affinity in a glycan-independent manner and that it bound selectively to the gp41 C-terminal heptad repeat. Native-PAGE, enzyme-linked immunosorbent assay, and CD spectroscopic analyses all revealed that retrocyclin-1 prevented 6-helix bundle formation. This mode of action, although novel for an innate effector molecule, resembles the mechanism of peptidic entry inhibitors based on portions of the gp41 sequence.

Binding Sites↗

PubMed2006

Peptide-lipid interactions of the beta-hairpin antimicrobial peptide tachyplesin and its linear derivatives from solid-state NMR.

The peptide-lipid interaction of a beta-hairpin antimicrobial peptide tachyplesin-1 (TP-1) and its linear derivatives are investigated to gain insight into the mechanism of antimicrobial activity. (31)P and (2)H NMR spectra of uniaxially aligned lipid bilayers of varying compositions and peptide concentrations are measured to determine the peptide-induced orientational disorder and the selectivity of membrane disruption by tachyplesin. The disulfide-linked TP-1 does not cause any disorder to the neutral POPC and POPC/cholesterol membranes but induces both micellization and random orientation distribution to the anionic POPE/POPG membranes above a peptide concentration of 2%. In comparison, the anionic POPC/POPG bilayer is completely unaffected by TP-1 binding, suggesting that TP-1 induces negative curvature strain to the membrane as a mechanism of its action. Removal of the disulfide bonds by substitution of Cys residues with Tyr and Ala abolishes the micellization of POPE/POPG bilayers but retains the orientation randomization of both POPC/POPG and POPE/POPG bilayers. Thus, linear tachyplesin derivatives have membrane disruptive abilities but use different mechanisms from the wild-type peptide. The different lipid-peptide interactions between TP-1 and other beta-hairpin antimicrobial peptides are discussed in terms of their molecular structure.

Antimicrobial Cationic Peptides↗

PubMed2006

Effective targeting of liposomes to liver and hepatocytes in vivo by incorporation of a Plasmodium amino acid sequence.

PURPOSE: Several species of the protozoan Plasmodium effectively target mammalian liver during the initial phase of host invasion. The purpose of this study was to demonstrate that a Plasmodium targeting amino acid sequence can be engineered into therapeutic nanoparticle delivery systems. METHODS: A 19-amino peptide from the circumsporozoite protein of Plasmodium berghei was prepared containing the conserved region I as well as a consensus heparan sulfate proteoglycan binding sequence. This peptide was attached to the distal end of a lipid-polyethylene glycol bioconjugate. The bioconjugate was incorporated into phosphatidylcholine liposomes containing fluorescently labeled lipids to follow blood clearance and organ distribution in vivo. RESULTS: When administered intravenously into mice, the peptide-containing liposomes were rapidly cleared from the circulation and were recovered almost entirely in the liver. Fluorescence and electron microscopy demonstrated that the liposomes were accumulated both by nonparenchymal cells and hepatocytes, with the majority of the liposomal material associated with hepatocytes. Accumulation of liposomes in the liver was several hundredfold higher compared to heart, lung, and kidney, and more than 10-fold higher compared to spleen. In liver slice experiments, liposome binding was specific to sites sensitive to heparinase. CONCLUSIONS: Incorporation of amino acid sequences that recognize glycosaminoglycans is an effective strategy for the development of targeted drug delivery systems.

Animals↗

PubMed2006

Orientation of a beta-hairpin antimicrobial peptide in lipid bilayers from two-dimensional dipolar chemical-shift correlation NMR.

The orientation of a beta-sheet membrane peptide in lipid bilayers is determined, for the first time, using two-dimensional (2D) (15)N solid-state NMR. Retrocyclin-2 is a disulfide-stabilized cyclic beta-hairpin peptide with antibacterial and antiviral activities. We used 2D separated local field spectroscopy correlating (15)N-(1)H dipolar coupling with (15)N chemical shift to determine the orientation of multiply (15)N-labeled retrocyclin-2 in uniaxially aligned phosphocholine bilayers. Calculated 2D spectra exhibit characteristic resonance patterns that are sensitive to both the tilt of the beta-strand axis and the rotation of the beta-sheet plane from the bilayer normal and that yield resonance assignment without the need for singly labeled samples. Retrocyclin-2 adopts a transmembrane orientation in dilauroylphosphatidylcholine bilayers, with the strand axis tilted at 20 degrees +/- 10 degrees from the bilayer normal, but changes to a more in-plane orientation in thicker 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidyl-choline (POPC) bilayers with a tilt angle of 65 degrees +/- 15 degrees . These indicate that hydrophobic mismatch regulates the peptide orientation. The 2D spectra are sensitive not only to the peptide orientation but also to its backbone (phi, psi) angles. Neither a bent hairpin conformation, which is populated in solution, nor an ideal beta-hairpin with uniform (phi, psi) angles and coplanar strands, agrees with the experimental spectrum. Thus, membrane binding orders the retrocyclin conformation by reducing the beta-sheet curvature but does not make it ideal. (31)P NMR spectra of lipid bilayers with different compositions indicate that retrocyclin-2 selectively disrupts the orientational order of anionic membranes while leaving zwitteronic membranes intact. These structural results provide insights into the mechanism of action of this beta-hairpin antimicrobial peptide.

Antimicrobial Cationic Peptides↗

PubMed2005

Intermolecular packing and alignment in an ordered beta-hairpin antimicrobial peptide aggregate from 2D solid-state NMR.

The aggregation and packing of a membrane-disruptive beta-hairpin antimicrobial peptide, protegrin-1 (PG-1), in the solid state are investigated to understand its oligomerization and hydrogen-bonding propensity. Incubation of PG-1 in phosphate buffer saline produced well-ordered nanometer-scale aggregates, as indicated by 13C and 15N NMR line widths, chemical shifts, and electron microscopy. Two-dimensional 13C and 1H spin diffusion experiments using C-terminus strand and N-terminus strand labeled peptides indicate that the beta-hairpin molecules in these ordered aggregates are oriented parallel to each other with like strands lining the intermolecular interface. In comparison, disordered and lyophilized peptide samples are randomly packed with both parallel and antiparallel alignments. The PG-1 aggregates show significant immobilization of the Phe ring near the beta-turn, further supporting the structural ordering. The intermolecular packing of PG-1 found in the solid state is consistent with its oligomerization in lipid bilayers. This solid-state aggregation approach may be useful for determining the quaternary structure of peptides in general and for gaining insights into the oligomerization of antimicrobial peptides in lipid bilayers in particular.

Algorithms↗

PubMed2005

Membrane-disruptive abilities of beta-hairpin antimicrobial peptides correlate with conformation and activity: a 31P and 1H NMR study.

The membrane interaction and solution conformation of two mutants of the beta-hairpin antimicrobial peptide, protegrin-1 (PG-1), are investigated to understand the structural determinants of antimicrobial potency. One mutant, [A(6,8,13,15)] PG-1, does not have the two disulfide bonds in wild-type PG-1, while the other, [Delta(4,18) G10] PG-1, has only half the number of cationic residues. 31P solid-state NMR lineshapes of uniaxially aligned membranes indicate that the membrane disorder induced by the three peptides decreases in the order of PG-1 > [Delta(4,18) G10] PG-1>>[A(6,8,13,15)] PG-1. Solution NMR studies of the two mutant peptides indicate that [Delta(4,18) G10] PG-1 preserves the beta-hairpin fold of the wild-type peptide while [A(6,8,13,15)] PG-1 adopts a random coil conformation. These NMR results correlate well with the known activities of these peptides. Thus, for this class of peptides, the presence of a beta-hairpin fold is more essential than the number of cationic charges for antimicrobial activity. This study indicates that 31P NMR lineshapes of uniaxially aligned membranes are well correlated with antimicrobial activity, and can be used as a diagnostic tool to understand the peptide-lipid interactions of these antimicrobial peptides.

Amino Acid Sequence↗

PubMed2005

Carbohydrate-binding molecules inhibit viral fusion and entry by crosslinking membrane glycoproteins.

Defensins are peptides that protect the host against microorganisms. Here we show that the theta-defensin retrocyclin 2 (RC2) inhibited influenza virus infection by blocking membrane fusion mediated by the viral hemagglutinin. RC2 was effective even after hemagglutinin attained a fusogenic conformation or had induced membrane hemifusion. RC2, a multivalent lectin, prevented hemagglutinin-mediated fusion by erecting a network of crosslinked and immobilized surface glycoproteins. RC2 also inhibited fusion mediated by Sindbis virus and baculovirus. Human beta-defensin 3 and mannan-binding lectin also blocked viral fusion by creating a protective barricade of immobilized surface proteins. This general mechanism might explain the broad-spectrum antiviral activity of many multivalent lectins of the innate immune system.

Anti-Infective Agents↗

PubMed2005

The N-terminus of hepcidin is essential for its interaction with ferroportin: structure-function study.

Hepcidin is the principal iron-regulatory hormone. It acts by binding to the iron exporter ferroportin, inducing its internalization and degradation, thereby blocking cellular iron efflux. The bioactive 25 amino acid (aa) peptide has a hairpin structure stabilized by 4 disulfide bonds. We synthesized a series of hepcidin derivatives and determined their bioactivity in a cell line expressing ferroportin-GFP fusion protein, by measuring the degradation of ferroportin-GFP and the accumulation of ferritin after peptide treatment. Bioactivity was also assayed in mice by the induction of hypoferremia. Serial deletion of N-terminal amino acids caused progressive decrease in activity which was completely lost when 5 N-terminal aa's were deleted. Synthetic 3-aa and 6-aa N-terminal peptides alone, however, did not internalize ferroportin and did not interfere with ferroportin internalization by native hepcidin. Deletion of 2 C-terminal aa's did not affect peptide activity. Removal of individual disulfide bonds by pairwise substitution of cysteines with alanines also did not affect peptide activity in vitro. However, these peptides were less active in vivo, likely because of their decreased stability in circulation. G71D and K83R, substitutions previously described in humans, did not affect hepcidin activity. Apart from the essential nature of the N-terminus, hepcidin structure appears permissive for mutations.

Amino Acid Sequence↗

PubMed2005

Determination of peptide oligomerization in lipid bilayers using 19F spin diffusion NMR.

Aggregation or oligomerization is important for the function of many membrane peptides such as ion channels and antimicrobial peptides. However, direct proof of aggregation and the determination of the number of molecules in the aggregate have been difficult due to the lack of suitable high-resolution methods for membrane peptides. We propose a 19F spin diffusion magic-angle-spinning NMR technique to determine the oligomeric state of peptides bound to the lipid bilayer. Magnetization transfer between chemically equivalent but orientationally different 19F spins on different molecules reduces the 19F magnetization in an exchange experiment. At long mixing times, the equilibrium 19F magnetization is 1/M, where M is the number of orientationally different molecules in the aggregate. The use of the 19F spin increases the homonuclear dipolar coupling and thus the distance reach. We demonstrate this technique on crystalline model compounds with known numbers of molecules in the asymmetric unit cell, and show that 19F spin diffusion is more efficient than that of 13C by a factor of approximately 500. Application to a beta-hairpin antimicrobial peptide, protegrin-1, shows that the peptide is almost completely dimerized in POPC bilayers at a concentration of 7.4 mol %. Decreasing the peptide concentration reduced the dimer fraction. Using a monomer-dimer equilibrium model, we estimate the DeltaG for dimer formation to be -10.2 +/- 2.3 kJ/mol. This is in good agreement with the previously measured free energy reduction for partitioning and aggregating beta-sheet peptides into phospholipid membranes. This 19F spin diffusion technique opens the possibility of determining the oligomeric structures of membrane peptides.

Anti-Infective Agents↗