Peptides In Lipid Bilayers Research Articles

Chain-Length Dependence of Peptide-Lipid Bilayer Interaction Strength and Binding Kinetics: A Combined Theoretical and Experimental Approach.

Physical interactions between polypeptide chains and lipid membranes underlie critical cellular processes. Yet, despite fundamental importance, key mechanistic aspects of these interactions remain elusive. Bulk experiments have revealed a linear relationship between free energy and peptide chain length in a model system, but does this linearity extend to the interaction strength and to the kinetics of lipid binding? To address these questions, we utilized a combination of coarse-grained molecular dynamics (CG MD) simulations, analytical modeling, and atomic force microscopy (AFM)-based single molecule force spectroscopy. Following previous bulk experiments, we focused on interactions between short hydrophobic peptides (WLn, n = 1, ..., 5) with 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) bilayers, a simple system that probes peptide primary structure effects. Potentials of mean force extracted from CG MD recapitulated the linearity of free energy with the chain length. Simulation results were quantitatively connected to bulk biochemical experiments via a single scaling factor of order unity, corroborating the methodology. Additionally, CG MD revealed an increase in the distance to the transition state, a result that weakens the dependence of the dissociation force on the peptide chain length. AFM experiments elucidated rupture force distributions and, through modeling, intrinsic dissociation rates. Taken together, the analysis indicates a rupture force plateau in the WLn-POPC system, suggesting that the final rupture event involves the last 2 or 3 residues. In contrast, the linear dependence on chain length was preserved in the intrinsic dissociation rate. This study advances the understanding of peptide-lipid interactions and provides potentially useful insights for the design of peptides with tailored membrane-interacting properties.

Simulation of oriented NMR spectra: Combining molecular dynamics and chemical shift tensor calculations.

Solid state NMR is widely used to study the orientation and other structural features of proteins and peptides in lipid bilayers. Using data obtained by PISEMA (Polarization Inversion Spin Exchange at Magic Angle) experiments, periodic spectral patterns arise from well-aligned α-helical molecules. Significant problems in the interpretation of PISEMA spectra may arise for systems that do not form perfectly defined secondary structures, like α-helices, or the signal pattern is disturbed by molecular motion. Here, we present a new method that combines molecular dynamics simulation with tensorial orientational constraints (MDOC) and chemical shift tensor calculations for the simulation and interpretation of PISEMA-like spectra. The calculations include the spectra arising from non α-helical molecules and molecules with non-uniform intrinsic mobility. In a first step, dipolar or quadrupolar interaction tensors drive molecular rotations and reorientations to obtain the proper mean values as observed in corresponding NMR experiments. In a second step, the coordinate snapshots of the MDOC simulations are geometry optimized with the isotropic 15 N chemical shifts as constraints using Bond Polarization Theory (BPT) to provide reliable 15 N CS tensor data. The averaged dipolar 1 H-15 N couplings and the δzz tensor components can then be combined to simulate PISEMA patterns. We apply this method to the ß-helical peptide gramicidin A (gA) and demonstrate that this method enables the assignment of most PISEMA resonances. In addition, MDOC simulations provide local order parameters for the calculated sites. These local order parameters reveal large differences in backbone mobility between L- and D-amino acids of gA.

Organizations of melittin peptides after spontaneous penetration into cell membranes

The antimicrobial peptide, melittin, is a potential next-generation antibiotic because melittin can spontaneously form pores in bacterial cell membranes and cause cytoplasm leakage. However, the organizations of melittin peptides in cell membranes remain elusive, which impedes the understanding of the poration mechanism. In this work, we use coarse-grained and all-atom molecular dynamics (MD) simulations to investigate the organizations of melittin peptides during and after spontaneous penetration into DPPC/POPG lipid bilayers. We find that the peptides in lipid bilayers adopt either a transmembrane conformation or a U-shaped conformation, which are referred to as T- and U-peptides, respectively. Several U-peptides and/or T-peptides aggregate to form stable pores. We analyze a T-pore consisting of four T-peptides and a U-pore consisting of three U-peptides and one T-peptide. In both pores, peptides are organized in a manner such that polar residues face inward and hydrophobic residues face outward, which stabilizes the pores and produces water channels. Compared with the U-pore, the T-pore has lower energy, larger pore diameter, and higher permeability. However, the T-pore occurs less frequently than the U-pore in our simulations, probably because the formation of the T-pore is kinetically slower than the U-pore. The stability and permeability of both pores are confirmed by 300 ns all-atom MD simulations. The peptide organizations obtained in this work should deepen the understanding of the stability, poration mechanism, and permeability of melittin, and facilitate the optimization of melittin to enhance the antibacterial ability.

Concentration-Dependent Effects of Cholesterol on the Dimerization of Amyloid-β Peptides in Lipid Bilayers.

Membrane disruption mediated by the accumulation of amyloid-β (Aβ) on cell membranes is central to the pathogenesis of Alzheimer's disease (AD). Cholesterol, an important component of membranes, is well-recognized as a risk factor in AD. It can affect the aggregation and pore formation of Aβ on membranes whereas the specific effects are rather complex, particularly regarding the non-linear response to cholesterol concentrations. Yet, the mechanistic understanding of the role of cholesterol in Aβ-membrane interactions remains incomplete. Herein, we employed microsecond-scale molecular dynamics simulations to investigate the effects of cholesterol on Aβ dimerization in a lipid bilayer containing different molar ratios of cholesterol (0, 20, and 40 mol %). Cholesterol reduces the time required for the formation of stable dimers and exerts dual effects on Aβ-membrane interactions. First, cholesterol promotes the extraction of the C-terminal region from the membrane to water. Consequently, at the ratios of 0 and 20 mol %, peptides are anchored at the membrane-water interface, but they are repelled to water at a ratio of 40 mol % with high structural flexibility. Second, cholesterol weakens Aβ-membrane interactions, thereby enhancing inter-peptide interactions. The former is favorable for dimerization while the latter is not. The balance between two factors eventually leads to a non-monotonic effect on the degree of dimerization, whereby the number of inter-peptide contacts is the largest at a cholesterol ratio of 20 mol %. These results provide atomistic insights into the regulation mechanism of Aβ42 aggregation by cholesterol and help to understand the pathological link between cholesterol and AD.

Clustering of tetrameric influenza M2 peptides in lipid bilayers investigated by 19F solid-state NMR

The influenza M2 protein forms a drug-targeted tetrameric proton channel to mediate virus uncoating, and carries out membrane scission to enable virus release. While the proton channel function of M2 has been extensively studied, the mechanism by which M2 catalyzes membrane scission is still not well understood. Previous fluorescence and electron microscopy studies indicated that M2 tetramers concentrate at the neck of the budding virus in the host plasma membrane. However, molecular evidence for this clustering is scarce. Here, we use 19F solid-state NMR to investigate M2 clustering in phospholipid bilayers. By mixing equimolar amounts of 4F-Phe47 labeled M2 peptide and CF3-Phe47 labeled M2 peptide and measuring F-CF3 cross peaks in 2D 19F19F correlation spectra, we show that M2 tetramers form nanometer-scale clusters in lipid bilayers. This clustering is stronger in cholesterol-containing membranes and phosphatidylethanolamine (PE) membranes than in cholesterol-free phosphatidylcholine and phosphatidylglycerol membranes. The observed correlation peaks indicate that Phe47 sidechains from different tetramers are less than ~2 nm apart. 1H19F correlation peaks between lipid chain protons and fluorinated Phe47 indicate that Phe47 is more deeply inserted into the lipid bilayer in the presence of cholesterol than in its absence, suggesting that Phe47 preferentially interacts with cholesterol. Static 31P NMR spectra indicate that M2 induces negative Gaussian curvature in the PE membrane. These results suggest that M2 tetramers cluster at cholesterol- and PE-rich regions of cell membranes to cause membrane curvature, which in turn can facilitate membrane scission in the last step of virus budding and release.

Tentative Peptide‒Lipid Bilayer Models Elucidating Molecular Behaviors and Interactions Driving Passive Cellular Uptake of Collagen-Derived Small Peptides.

Collagen contains hydroxyproline (Hyp), which is a unique amino acid. Three collagen-derived small peptides (Gly-Pro-Hyp, Pro-Hyp, and Gly-Hyp) interacting across a lipid bilayer (POPC model membrane) for cellular uptakes of these collagen-derived small peptides were studied using accelerated molecular dynamics simulation. The ligands were investigated for their binding modes, hydrogen bonds in each coordinate frame, and mean square displacement (MSD) in the Z direction. The lipid bilayers were evaluated for mass and electron density profiles of the lipid molecules, surface area of the head groups, and root mean square deviation (RMSD). The simulation results show that hydrogen bonding between the small collagen peptides and plasma membrane plays a significant role in their internalization. The translocation of the small collagen peptides across the cell membranes was shown. Pro-Hyp laterally condensed the membrane, resulting in an increase in the bilayer thickness and rigidity. Perception regarding molecular behaviors of collagen-derived peptides within the cell membrane, including their interactions, provides the novel design of specific bioactive collagen peptides for their applications.

Heterodimer and pore formation of magainin 2 and PGLa: The anchoring and tilting of peptides in lipid bilayers

Mixtures of Magainin 2 and PGLa are simulated with 94 nm-sized bilayers composed of phospholipids and lyso-phospholipids for 3 μs using coarse-grained force fields. Calculation of the bilayer bending modulus shows that bilayers become more flexible in the presence of lyso-lipids or peptides, in agreement with experiments. Starting with the initial configuration of peptides randomly distributed on the bilayer surface, peptides aggregate, insert to the bilayer, and form pores. Aggregated peptides do not retain side-by-side heterodimeric structure but instead show the anchoring between C-terminal groups of magainin 2 and PGLa, which allows the deeper insertion of PGLa into the bilayer. In particular, due to the anchoring of magainin 2 and PGLa, the deeply inserted PGLa pull magainin 2 into contact with the edge of the opposite leaflet of the bilayer, which stabilizes the pore. In addition to these biophysical insights, anionic unsaturated-phospholipid bilayers are also simulated to mimic bacterial cell membranes, showing less extent of PGLa insertion and no pore formation. These simulation findings indicate that these synergistic heterodimers have the anchoring structure rather than the side-by-side structure, which supports the experimental observations suggesting the deeper insertion of PGLa and pore formation via the anchoring between anionic C-terminus of magainin 2 and cationic C-terminus of PGLa.

Interfacial Behaviors of Transmembrane Peptides in Lipid Bilayer Studied by X-Ray and Neutron Reflectivity

Lipids and proteins can influence each other in so many different ways. Lipids may affect the structure of membrane proteins by influencing their backbone conformation, the tilt, rotation angles of their transmembrane (TM) segments, and the orientation of their side chains. The membrane-spanning parts in integral membrane proteins are predominantly hydrophobic, and most often helical. At the lipid-protein interface, the TM part of the protein and the hydrocarbon chains of the lipid molecules have to coexist to maintain the integrity of the membrane. Lipids are important components of lipid membrane are used in various experiments reported in this thesis and can act as model lipid bilayers. Once they support on solid substrate like silicon wafers, their structural properties can investigate by X-ray and neutron reflectivity and by other useful techniques. Reflectivity technique can provide detailed information such as their thickness and interaction between lipids and peptides. The thesis reports a detailed investigation of these lipids and peptides by X-ray and neutron reflection techniques

Conformational Dynamics and Energetics of Melittin and its Diastereomer Interacting with POPC and POPG Lipid Bilayers: A Molecular Dynamics Study

Often amphipathic membrane-active peptides assume helical secondary structure when partitioning into lipid membranes. Characterizing this membrane-induced folding process at single molecule level is fundamental to understanding peptide lipid-membrane interactions. To this end, we use all-atom, long time scale (∼1µs) molecular dynamics (MD) simulations to investigate and compare the conformational dynamics and energetics of the well-studied peptide melittin (MWT) and one of its less-studied diastereomer, with four D-amino acids, (MD4) interacting respectively with a zwitterionic POPC and an anionic POPG lipid bilayer. Unlike in previous MD studies, we start from a completely folded conformation of the peptides and follow their unfolding through free MD simulations. We find that (i) both MWT and MD4 unfold in solution and remain mostly disordered; (ii) MWT binds to both POPC and POPG, remaining mostly folded (with more than 70% helical content); and (iii) MD4 binds only to POPG and it is mostly disordered (with less than 40% helical content). Furthermore, by calculating the potential of mean force, as a function of peptide-lipid bilayer separation, we have determined the relative dissociation energy of MWT and MD4 from POPG, which can be related to the membrane induced folding energy of melittin. To our knowledge, this is the first MD study of the MD4 melittin analog, and the obtained results are in good agreement with previous experimental studies. Work supported in part by a grant from the MU Research Board. The computation for this work was performed on the high HPC infrastructure provided by RCSS and in part by the NSF under grant number CNS-1429294 at the University of Missouri, Columbia MO.

Characterization of apolipoprotein A-I peptide phospholipid interaction and its effect on HDL nanodisc assembly.

Background: Synthetic HDLs (sHDLs), small nanodiscs of apolipoprotein mimetic peptides surrounding lipid bilayers, were developed clinically for atheroma regression in cardiovascular patients. Formation of HDL involves interaction of apolipoprotein A-I (ApoA-I) with phospholipid bilayers and assembly into lipid-protein nanodiscs.Purpose: The objective of this study is to improve understanding of physico-chemical aspects of HDL biogenesis such as the thermodynamics of ApoA-I-peptide membrane insertion, lipid binding, and HDL self-assembly to improve our ability to form homogeneous sHDL nanodiscs that are suitable for clinical administration.Methods: The ApoA-I-mimetic peptide, 22A, was combined with either egg sphingomyelin (eSM) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) phospholipid vesicles to form sHDL. The sHDL assembly process was investigated through lipid vehicle solubilization assays and characterization of purity, size, and morphology of resulting nanoparticles via gel permeation chromatography (GPC), dynamic light scattering (DLS), and transmission electron microscopy (TEM). Peptide-lipid interactions involved were further probed by sum frequency generation (SFG) vibrational spectroscopy and attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR). The pharmacokinetics of eSM-sHDL and POPC-sHDL nanodiscs were investigated in Sprague Dawley rats.Results: sHDL formation was temperature-dependent, with spontaneous formation of sHDL nanoparticles occurring only at temperatures exceeding lipid transition temperatures as evidenced by DLS, GPC, and TEM characterization. SFG and ATR-FTIR spectroscopy findings support a change in peptide-lipid bilayer interactions at temperatures above the lipid transition temperature. Lipid-22A interactions were stronger with eSM than with POPC, which resulted in the formation of more homogeneous sHDL nanoparticles with longer in vivo circulation time as evidenced the PK study.Conclusion: Physico-chemical characteristics of sHDL are in part determined by phospholipid composition. Optimization of phospholipid composition may be utilized to improve the stability and homogeneity of sHDL.

Dimerization in tailoring uptake efficacy of the HSV-1 derived membranotropic peptide gH625

gH625 constitutes a promising delivery vehicle for the transport of therapeutic biomacromolecules across membrane barriers. We report an application of multivalency to create a complex nanosystem for delivery and to elucidate the mechanism of peptide-lipid bilayer interactions. Multivalency may offer a route to enhance gH625 cellular uptake as demonstrated by results obtained on dimers of gH625 by fluorescence spectroscopy, circular dichroism, and surface plasmon resonance. Moreover, using both phase contrast and light sheet fluorescence microscopy we were able to characterize and visualize for the first time the fusion of giant unilamellar vesicles caused by a membranotropic peptide.

Methodology for identification of pore forming antimicrobial peptides from soy protein subunits β-conglycinin and glycinin

Antimicrobial peptides (AMPs) inactivate microbial cells through pore formation in cell membrane. Because of their different mode of action compared to antibiotics, AMPs can be effectively used to combat drug resistant bacteria in human health. AMPs can also be used to replace antibiotics in animal feed and immobilized on food packaging films. In this research, we developed a methodology based on mechanistic evaluation of peptide-lipid bilayer interaction to identify AMPs from soy protein. Production of AMPs from soy protein is an attractive, cost-saving alternative for commercial consideration, because soy protein is an abundant and common protein resource. This methodology is also applicable for identification of AMPs from any protein. Initial screening of peptide segments from soy glycinin (11S) and soy β-conglycinin (7S) subunits was based on their hydrophobicity, hydrophobic moment and net charge. Delicate balance between hydrophilic and hydrophobic interactions is necessary for pore formation. High hydrophobicity decreases the peptide solubility in aqueous phase whereas high hydrophilicity limits binding of the peptide to the bilayer. Out of several candidates chosen from the initial screening, two peptides satisfied the criteria for antimicrobial activity, viz. (i) lipid-peptide binding in surface state and (ii) pore formation in transmembrane state of the aggregate. This method of identification of antimicrobial activity via molecular dynamics simulation was shown to be robust in that it is insensitive to the number of peptides employed in the simulation, initial peptide structure and force field. Their antimicrobial activity against Listeria monocytogenes and Escherichia coli was further confirmed by spot-on-lawn test.

The effect of orientation dynamics in melittin as antimicrobial peptide in lipid bilayer calculated by free energy method

Melittin is a widely studied antimicrobial peptide which has gained interest because of its potential in therapeutic use. To perform its antimicrobial function, the melittin prefer to be in a certain state of membrane. In this study, we simulate the melittin in horizontal orientation towards the surface of membrane and in vertical orientation inside transmembrane state. The free energies are then calculated to asses the most favorable orientation, indicating its stable structure, of this peptide interacting with the lipid bilayer. Umbrella sampling with and without pulling simulations is carried out to obtain the sampling windows which used in observing the free energy changes along the reaction coordinate. From our simulations, we found that melittin in vertical orientation in transmembrane state has smaller free energy value than the horizontal one indicating that melittin is more favorable in this configuration. Our results show that melittin undergoes reorientation process from horizontal to vertical to perform its antimicrobial function.

Chemical synthesis of transmembrane peptide and its application for research on the transmembrane-juxtamembrane region of membrane protein.

Membrane proteins possess one or more hydrophobic regions that span the membrane and interact with the lipids that constitute the membrane. The interactions between the transmembrane (TM) region and lipids affect the structure and function of these membrane proteins. Molecular characterization of synthetic TM peptides in lipid bilayers helps to understand how the TM region participates in the formation of the structure and in the function of membrane proteins. The use of synthetic peptides enables site-specific labeling and modification and allows for designing of an artificial TM sequence. Research involving such samples has resulted in significant increase in the knowledge of the mechanisms that govern membrane biology. In this review, the chemical synthesis of TM peptides has been discussed. The preparation of synthetic TM peptides is still not trivial; however, the accumulated knowledge summarized here should provide a basis for preparing samples for spectroscopic analyses. The application of synthetic TM peptides for gaining insights into the mechanism of signal transduction by receptor tyrosine kinase (RTK) has also been discussed. RTK is a single TM protein and is one of the difficult targets in structural biology as crystallization of the full-length receptor has not been successful. This review describes the structural characterization of the synthetic TM-juxtamembrane sequence and proposes a possible scheme for the structural changes in this region for the activation of ErbBs, the epidermal growth factor receptor family. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 613-621, 2016.

Effects of dipole modifiers on channel-forming activity of amyloid and amyloid-like peptides in lipid bilayers

We have studied the steady-state transmembrane current induced by amyloid and amyloid-like peptides in lipid bilayers in the presence of dipole modifiers. It has been shown that the addition of dipole modifier, phloretin, to the membrane bathing solutions leads to an increase in the multichannel activity of amyloid beta-peptide fragment 25-35, [Gly35]-amyloid beta-peptide fragment 25--35, prion protein fragment 106-126 and amyloid-like peptides myr-BASP1 (1--13), myr-BASP1(1--19) and GAP-43(1--40). We have found that the effect of phloretin is not the result of dipole potential changes due to adsorption of this modifier on the membrane. Using the various fragments of amyloid beta-peptide, presenilin, prion protein and neuronal proteins BASP1 and GAP-43 allowes to conclude that the steady-state peptide-induced transmembrane current in the case of addition of phloretin is due to the electrostatic interaction between the positively charged channel-forming agents and negatively charged dipole modifier. The results obtained by electron microscopy have demonstrated that this interaction increases degree of peptide oligomerization.

Analysis of Piscidin and Lipopolysaccharide Interactions: A Step Towards Characterizing Immunomodulation

Lipopolysaccharide (LPS) is one of the most extensively studied pathogen-associated molecular patterns (PAMPs), as it composes 90% of the membrane in Gram-negative bacteria. Recognition of micellar aggregates of LPS by the immune system subsequently mounts an inflammatory response against infection. However, if this process is over exaggerated, high levels of cytokines may become detrimental, leading to organ shutdown and septic shock. Various cationic antimicrobial peptides (AMPs) have been shown to decrease the transcription and release of pro-inflammatory cytokines associated with LPS recognition.Piscidin is a cationic antimicrobial peptide first isolated in the mast cells of fish. In the presence of a lipid bilayer, it folds into an amphipathic α-helix structure, which facilitates peptide-lipid bilayer binding. Based on the studies of other cationic AMPs, it was hypothesized that piscidin-1 (P1) and piscidin-3 (P3) would fold into their α-helical state and bind strongly to LPS, breaking apart its large aggregates. Using isothermal calorimetry to explore the thermodynamics of binding, it was shown that both peptides bound strongly to LPS through an exothermic reaction. Furthermore, fluorescence-dequenching studies were carried out using Fluorescein isothiocyanate labeled LPS (FITC-LPS), demonstrating piscidin's ability to separate FITC-LPS aggregates. Finally, both peptides were induced by LPS to be approximately 97% helical from their native random coil state, as shown by circular dichroism. Based upon these results, it is clear that significant interactions occur between LPS and piscidin, warranting future exploration of the effect of piscidin on LPS recognition by immune cells.

Interaction analysis of a ladder-shaped polycyclic ether and model transmembrane peptides in lipid bilayers by using Förster resonance energy transfer and polarized attenuated total reflection infrared spectroscopy

Ladder-shaped polycyclic ethers (LSPs) are predicted to interact with membrane proteins; however, the underlying mechanism has not been satisfactorily elucidated. It has been hypothesized that LSPs possess non-specific affinity to α-helical segments of transmembrane proteins. To verify this hypothesis, we constructed a model LSP interaction system in a lipid bilayer. We prepared 5 types of α-helical peptides and reconstituted them in liposomes. The reconstitution and orientation of these peptides in the liposomes were examined using polarized attenuated total reflection infrared (ATR-IR) spectroscopy and gel filtration. The results revealed that 4 peptides were retained in liposomes, and 3 of them formed stable transmembrane structures. The interaction between the LSP and the peptides was investigated using Förster resonance energy transfer (FRET). In the lipid bilayer, the LSP strongly recognized the peptides that possessed aligned hydrogen donating groups with leucine caps. We propose that this leucine-capped 16-amino acid sequence is a potential LPS binding motif.

Structural Plasticity in the Topology of the Membrane-Interacting Domain of HIV-1 gp41

We use a number of computational and experimental approaches to investigate the membrane topology of the membrane-interacting C-terminal domain of the HIV-1 gp41 fusion protein. Several putative transmembrane regions are identified using hydrophobicity analysis based on the Wimley-White scales, including the membrane-proximal external region (MPER). The MPER region is an important target for neutralizing anti-HIV monoclonal antibodies and is believed to have an interfacial topology in the membrane. To assess the possibility of a transmembrane topology of MPER, we examined the membrane interactions of a peptide corresponding to a 22-residue stretch of the MPER sequence (residues 662–683) using fluorescence spectroscopy and oriented circular dichroism. In addition to the previously reported interfacial location, we identify a stable transmembrane conformation of the peptide in synthetic lipid bilayers. All-atom molecular dynamics simulations of the MPER-derived peptide in a lipid bilayer demonstrate a stable helical structure with an average tilt of 24 degrees, with the five tryptophan residues sampling different environments inside the hydrocarbon core of the lipid bilayer, consistent with the observed spectral properties of intrinsic fluorescence. The degree of lipid bilayer penetration obtained by computer simulation was verified using depth-dependent fluorescence quenching of a selectively attached fluorescence probe. Overall, our data indicate that the MPER sequence can have at least two stable conformations in the lipid bilayer, interfacial and transmembrane, and suggest a possibility that external perturbations can switch the topology during physiological functioning.

Structural Plasticity in the Topology of Membrane-Spanning Domain of HIV-1 gp41

We use a number of computational and experimental approaches to investigate the mem-brane topology of the Membrane-Spanning Domain of the HIV-1 gp41 ectodomain. Several new putative transmembrane regions are identified using hydrophobicity analysis based on Wimley-White scales, including the membrane-proximal external region (MPER). The MPER region is an important target for neutralizing anti-HIV monoclonal antibodies and is believed to have an interfacial topology in the membrane. To assess the possibility of a transmembrane topology of MPER we examined the membrane interactions of a peptide corresponding to a 22 residue long stretch of MPER sequence (residues 662-683) using fluo-rescence spectroscopy and oriented circular dichroism. In addition to the previously report-ed interfacial location, we identify a stable transmembrane conformation of the peptide in synthetic lipid bilayers. All-atom molecular dynamics simulations of the MPER-derived peptide in a lipid bilayer demonstrate a stable helical structure with an average tilt of 24 de-grees with the five tryptophan residues sampling different environments inside bilayer's hy-drocarbon core, consistent with spectral properties of intrinsic fluorescence. The degree of lipid bilayer penetration obtained by computer simulation was verified using depth-dependent fluorescence quenching of selectively attached fluorescence probe. Our overall data indicate that the MPER sequence can have at least two stable conformations in the lipid bilayer, interfacial and transmembrane, and suggest a possibility that external perturbations can switch the topology during physiological functioning. Supported by NIH GM069783.

Lipid Tail Protrusion in Simulations Predicts Fusogenic Activity of Influenza Fusion Peptide Mutants and Conformational Models

Fusion peptides from influenza hemagglutinin act on membranes to promote membrane fusion, but the mechanism by which they do so remains unknown. Recent theoretical work has suggested that contact of protruding lipid tails may be an important feature of the transition state for membrane fusion. If this is so, then influenza fusion peptides would be expected to promote tail protrusion in proportion to the ability of the corresponding full-length hemagglutinin to drive lipid mixing in fusion assays. We have performed molecular dynamics simulations of influenza fusion peptides in lipid bilayers, comparing the X-31 influenza strain against a series of N-terminal mutants. As hypothesized, the probability of lipid tail protrusion correlates well with the lipid mixing rate induced by each mutant. This supports the conclusion that tail protrusion is important to the transition state for fusion. Furthermore, it suggests that tail protrusion can be used to examine how fusion peptides might interact with membranes to promote fusion. Previous models for native influenza fusion peptide structure in membranes include a kinked helix, a straight helix, and a helical hairpin. Our simulations visit each of these conformations. Thus, the free energy differences between each are likely low enough that specifics of the membrane environment and peptide construct may be sufficient to modulate the equilibrium between them. However, the kinked helix promotes lipid tail protrusion in our simulations much more strongly than the other two structures. We therefore predict that the kinked helix is the most fusogenic of these three conformations.