Promote Membrane Fusion Research Articles

Mitochondrial Membrane Fusion: Computational Modeling of Mitofusins

Fzo1, a large GTPase of the Dynamin-Related Proteins superfamily, is a key component in mitochondrial outer membrane fusion and is required for maintaining mitochondrial dynamics and morphology. The protein is anchored to the outer membrane by two transmembrane segments and its N-terminal GTPase domain and C-terminal are exposed to the cytosol. Recent data indicate that the GTPase domain of Fzo1 would induce a conformational change concomitant with mitochondrial tethering, thus promoting membrane fusion [1]. We investigate the structure and dynamics of Fzo1 through molecular modeling and all-atom simulation in a model mixed lipid bilayer, closely linked to experiments. Our structural model integrates information from several template structures, experimental knowledge, as well as ab initio models of the transmembrane segments that are unique to Fzo1. The model is validated experimentally through charge swap mutations across predicted salt bridges and a series of N-terminal truncation mutants indicates that this region is dispensable for function. Our approach unravels hinges domains involved in the conformational change and identified critical residues required for protein stability. Moreover several point mutation found to disrupt the architecture of the protein are located in the coiled-coil domain which has been shown fundamental for the protein [2]. Finally, we dissected key residues in protein-GDP interaction providing fundamental insights about molecular mechanisms by which mitofusins catalyze membrane fusion.[1] Cohen, M.M., Amiott, E.A., Day, A.R., Leboucher, G.P., Erin, N.P., Glickman, M.H., McCaffery, J.M, Shaw, J.M. and Weissman, A.M. (2011). J. Cell Sci. 124, 1403-1410.[2] Griffin, E. E. and Chan, D. C. (2006). J. Biol. Chem. 281, 16599-16606.

A non-zipper-like tetrameric coiled coil promotes membrane fusion

A parallel heterodimeric coiled coil can be mutated to an antiparallel tetrameric species by reversing the sequences of one of the peptides. This tetramer is capable of facilitating fast, efficient, membrane fusion of liposomes.

PH-Dependent Formation and Disintegration of the Influenza A Virus Protein Scaffold To Provide Tension for Membrane Fusion.

Influenza virus is taken up from a pH-neutral extracellular milieu into an endosome, whose contents then acidify, causing changes in the viral matrix protein (M1) that coats the inner monolayer of the viral lipid envelope. At a pH of ~6, M1 interacts with the viral ribonucleoprotein (RNP) in a putative priming stage; at this stage, the interactions of the M1 scaffold coating the lipid envelope are intact. The M1 coat disintegrates as acidification continues to a pH of ~5 to clear a physical path for the viral genome to transit from the viral interior to the cytoplasm. Here we investigated the physicochemical mechanism of M1's pH-dependent disintegration. In neutral media, the adsorption of M1 protein on the lipid bilayer was electrostatic in nature and reversible. The energy of the interaction of M1 molecules with each other in M1 dimers was about 10 times as weak as that of the interaction of M1 molecules with the lipid bilayer. Acidification drives conformational changes in M1 molecules due to changes in the M1 charge, leading to alterations in their electrostatic interactions. Dropping the pH from 7.1 to 6.0 did not disturb the M1 layer; dropping it lower partially desorbed M1 because of increased repulsion between M1 monomers still stuck to the membrane. Lipid vesicles coated with M1 demonstrated pH-dependent rupture of the vesicle membrane, presumably because of the tension generated by this repulsive force. Thus, the disruption of the vesicles coincident with M1 protein scaffold disintegration at pH 5 likely stretches the lipid membrane to the point of rupture, promoting fusion pore widening for RNP release. Influenza remains a top killer of human beings throughout the world, in part because of the influenza virus's rapid binding to cells and its uptake into compartments hidden from the immune system. To attack the influenza virus during this time of hiding, we need to understand the physical forces that allow the internalized virus to infect the cell. In particular, we need to know how the protective coat of protein inside the viral surface reacts to the changes in acid that come soon after internalization. We found that acid makes the molecules of the protein coat push each other while they are still stuck to the virus, so that they would like to rip the membrane apart. This ripping force is known to promote membrane fusion, the process by which infection actually occurs.

The Atomic Structure of the HIV-1 gp41 Transmembrane Domain and Its Connection to the Immunogenic Membrane-proximal External Region

The membrane-proximal external region (MPER) C-terminal segment and the transmembrane domain (TMD) of gp41 are involved in HIV-1 envelope glycoprotein-mediated fusion and modulation of immune responses during viral infection. However, the atomic structure of this functional region remains unsolved. Here, based on the high resolution NMR data obtained for peptides spanning the C-terminal segment of MPER and the TMD, we report two main findings: (i) the conformational variability of the TMD helix at a membrane-buried position; and (ii) the existence of an uninterrupted α-helix spanning MPER and the N-terminal region of the TMD. Thus, our structural data provide evidence for the bipartite organization of TMD predicted by previous molecular dynamics simulations and functional studies, but they do not support the breaking of the helix at Lys-683, as was suggested by some models to mark the initiation of the TMD anchor. Antibody binding energetics examined with isothermal titration calorimetry and humoral responses elicited in rabbits by peptide-based vaccines further support the relevance of a continuous MPER-TMD helix for immune recognition. We conclude that the transmembrane anchor of HIV-1 envelope is composed of two distinct subdomains: 1) an immunogenic helix at the N terminus also involved in promoting membrane fusion; and 2) an immunosuppressive helix at the C terminus, which might also contribute to the late stages of the fusion process. The unprecedented high resolution structural data reported here may guide future vaccine and inhibitor developments.

Vacuolar ATPase in Phagosome-Lysosome Fusion

The vacuolar H(+)-ATPase (v-ATPase) complex is instrumental in establishing and maintaining acidification of some cellular compartments, thereby ensuring their functionality. Recently it has been proposed that the transmembrane V0 sector of v-ATPase and its a-subunits promote membrane fusion in the endocytic and exocytic pathways independent of their acidification functions. Here, we tested if such a proton-pumping independent role of v-ATPase also applies to phagosome-lysosome fusion. Surprisingly, endo(lyso)somes in mouse embryonic fibroblasts lacking the V0 a3 subunit of the v-ATPase acidified normally, and endosome and lysosome marker proteins were recruited to phagosomes with similar kinetics in the presence or absence of the a3 subunit. Further experiments used macrophages with a knockdown of v-ATPase accessory protein 2 (ATP6AP2) expression, resulting in a strongly reduced level of the V0 sector of the v-ATPase. However, acidification appeared undisturbed, and fusion between latex bead-containing phagosomes and lysosomes, as analyzed by electron microscopy, was even slightly enhanced, as was killing of non-pathogenic bacteria by V0 mutant macrophages. Pharmacologically neutralized lysosome pH did not affect maturation of phagosomes in mouse embryonic cells or macrophages. Finally, locking the two large parts of the v-ATPase complex together by the drug saliphenylhalamide A did not inhibit in vitro and in cellulo fusion of phagosomes with lysosomes. Hence, our data do not suggest a fusion-promoting role of the v-ATPase in the formation of phagolysosomes.

Mitochondrial Genome Maintenance 1 (Mgm1) Protein Alters Membrane Topology and Promotes Local Membrane Bending

Large GTPases of the dynamin superfamily promote membrane fusion and division, processes that are crucial for intracellular trafficking and organellar dynamics. To promote membrane scission, dynamin proteins polymerize, wrap around, and constrict the membrane; however, the mechanism underlying their role in membrane fusion remains unclear. We previously reported that the mitochondrial dynamin-related protein mitochondrial genome maintenance 1 (Mgm1) mediates fusion by first tethering opposing membranes and then undergoing a nucleotide-dependent structural transition. However, it is still unclear how Mgm1 directly affects the membrane to drive fusion of tethered membranes. Here, we show that Mgm1 association with the membrane alters the topography of the membrane, promoting local membrane bending. We also demonstrate that Mgm1 creates membrane ruffles resulting in the formation of tubular structures on both supported lipid bilayers and liposomes. These data suggest that Mgm1 membrane interactions impose a mechanical force on the membrane to overcome the hydrophilic repulsion of the phospholipid head groups and initiate the fusion reaction. The work reported here provides new insights into a possible mechanism of Mgm1-driven mitochondrial membrane fusion and sheds light into how members of the dynamin superfamily function as fusion molecules.

Rho GTPases and the Downstream Effectors Actin-related Protein 2/3 (Arp2/3) Complex and Myosin II Induce Membrane Fusion at Self-contacts

Actin regulation is required for membrane activities that drive cell adhesion and migration. The Rho GTPase family plays critical roles in actin and membrane dynamics; however, the roles of the Rho GTPase family are not limited to cell adhesion and migration. Using micron-sized obstacles to induce the formation of self-contacts in epithelial cells, we previously showed that self-adhesion is distinct from cell-to-cell adhesion in that self-contacts are eliminated by membrane fusion. In the current study, we identified Rho GTPases, RhoA, Rac1, and Cdc42, as potential upstream regulators of membrane fusion. The RhoA downstream effector myosin II is required for fusion as the expression of mutant myosin light chain reduced membrane fusion. Furthermore, an inhibitor of the Arp2/3 complex, a downstream effector of Rac1 and Cdc42, also reduced self-contact-induced membrane fusion. At self-contacts, while the concentration of E-cadherin diminished, the intensity of GFP-tagged Arp3 rapidly fluctuated then decreased and stabilized after membrane fusion. Taken together, these data suggest that the Arp2/3 complex-mediated actin polymerization brings two opposing membranes into close apposition by possibly excluding E-cadherin from contact sites, thus promoting membrane fusion at self-contacts.

Temporally Resolving Protein and Lipid Colocalization at Exocytic Sites in INS-1 Cells

The controlled release of material from cells, called exocytosis, is critical for many life processes ranging from synaptic transmission to hormone secretion. Exocytosis is mediated by the assembly of SNARE proteins into a complex that links vesicular and plasma membranes and promotes membrane fusion. Despite a detailed understanding of SNARE complex assembly, a comprehensive description of the arrival and departure of the myriad of other proteins involved in exocytosis is lacking. Here, we describe the localization and dynamics of over a dozen proteins at sites of dense core vesicle exocytosis inside living INS-1 cells. We use two color total internal reflectance fluorescence microscopy to visualize transiently transfected, fluorescently tagged proteins. NPY-GFP, a dense core vesicle cargo protein, is used to localize vesicles and temporally align many exocytic events across several cells. mCherry is used to localize a particular exocytic protein of interest relative to the dense core vesicle and determine its temporal behavior relative to the moment of fusion. Intriguingly, we find that both positive and negative regulators of SNARE complex assembly appear to be present at exocytic sites and diffuse away after fusion occurs. This data suggests that effectors acting on the SNARE complex must be biochemically or conformationally regulated; they are not spatially regulated by exclusion or inclusion at exocytic sites. We are currently examining one exocytic protein, tomosyn, to determine how this protein might be regulated. We are also investigating lipid dynamics at the site of exocytosis using fluorescently tagged lipid binding domains. Our imaging approach should show whether critical lipids are locally synthesized or laterally diffuse to the site of exocytosis. With our data we seek to build a thorough description of protein and lipid behavior at the moment of exocytosis.

Recognition of Lipid Domain Boundaries by the HIV Fusion Peptide is an Essential Step for HIV Membrane Fusion

“Lipid rafts” which are cholesterol-enriched regions of the plasma membrane have been recognized as possible platforms for HIV entry. However, due to their highly dynamic and nanoscopic nature in biological membranes, it is challenging to investigate the role of lipid rafts in HIV-host interactions. In this study, we have used model systems with microscopic raft-like domains (Lo phase) in supported lipid bilayers and giant unilamellar vesicles mimicking HIV envelopes and T-cell membranes. We show that the phase separation of HIV or T-cell lipid mixtures is cholesterol-dependent, and membrane binding and lipid mixing are much more efficient in vesicles with coexisting Ld and Lo phases than those with single Ld or Lo phase, indicating that lipid phase separation is necessary and sufficient for efficient membrane fusion. Interestingly, time-resolved TIRF microscopy demonstrates that the HIV fusion peptide preferentially targets Lo/Ld boundary regions and promotes membrane fusion at the interface between Lo and Ld phases. Analysis of individual fusion events shows that pure Ld phase vesicles proceed to hemifusion and only vesicles with Lo/Ld phase boundaries fuse fully. Based on our designed minimal systems for understanding lipid raft-dependent HIV-host interactions, we propose that recognition of domain boundaries by the HIV fusion peptide is an essential step for HIV entry.

Sequential conformational rearrangements in flavivirus membrane fusion.

The West Nile Virus (WNV) envelope protein, E, promotes membrane fusion during viral cell entry by undergoing a low-pH triggered conformational reorganization. We have examined the mechanism of WNV fusion and sought evidence for potential intermediates during the conformational transition by following hemifusion of WNV virus-like particles (VLPs) in a single particle format. We have introduced specific mutations into E, to relate their influence on fusion kinetics to structural features of the protein. At the level of individual E subunits, trimer formation and membrane engagement of the threefold clustered fusion loops are rate-limiting. Hemifusion requires at least two adjacent trimers. Simulation of the kinetics indicates that availability of competent monomers within the contact zone between virus and target membrane makes trimerization a bottleneck in hemifusion. We discuss the implications of the model we have derived for mechanisms of membrane fusion in other contexts.

Sphingomyelin Synthase 2, but Not Sphingomyelin Synthase 1, Is Involved in HIV-1 Envelope-mediated Membrane Fusion

Membrane fusion between the viral envelope and plasma membranes of target cells has previously been correlated with HIV-1 infection. Lipids in the plasma membrane, including sphingomyelin, may be crucially involved in HIV-1 infection; however, the role of lipid-metabolic enzymes in membrane fusion remains unclear. In this study, we examined the roles of sphingomyelin synthase (SMS) in HIV-1 Env-mediated membrane fusion using a cell-cell fusion assay with HIV-1 mimetics and their target cells. We employed reconstituted cells as target cells that stably express Sms1 or Sms2 in Sms-deficient cells. Fusion susceptibility was ∼5-fold higher in Sms2-expressing cells (not in Sms1-expressing cells) than in Sms-deficient cells. The enhancement of fusion susceptibility observed in Sms2-expressing cells was reversed and reduced by Sms2 knockdown. We also found that catalytically nonactive Sms2 promoted membrane fusion susceptibility. Moreover, SMS2 co-localized and was constitutively associated with the HIV receptor·co-receptor complex in the plasma membrane. In addition, HIV-1 Env treatment resulted in a transient increase in nonreceptor tyrosine kinase (Pyk2) phosphorylation in Sms2-expressing and catalytically nonactive Sms2-expressing cells. We observed that F-actin polymerization in the region of membrane fusion was more prominent in Sms2-expressing cells than Sms-deficient cells. Taken together, our research provides insight into a novel function of SMS2 which is the regulation of HIV-1 Env-mediated membrane fusion via actin rearrangement.

A distinct tethering step is vital for vacuole membrane fusion

Past experiments with reconstituted proteoliposomes, employing assays that infer membrane fusion from fluorescent lipid dequenching, have suggested that vacuolar SNAREs alone suffice to catalyze membrane fusion in vitro. While we could replicate these results, we detected very little fusion with the more rigorous assay of lumenal compartment mixing. Exploring the discrepancies between lipid-dequenching and content-mixing assays, we surprisingly found that the disposition of the fluorescent lipids with respect to SNAREs had a striking effect. Without other proteins, the association of SNAREs in trans causes lipid dequenching that cannot be ascribed to fusion or hemifusion. Tethering of the SNARE-bearing proteoliposomes was required for efficient lumenal compartment mixing. While the physiological HOPS tethering complex caused a few-fold increase of trans-SNARE association, the rate of content mixing increased more than 100-fold. Thus tethering has a role in promoting membrane fusion that extends beyond simply increasing the amount of total trans-SNARE complex.

Membrane Dynamics and Lipid Interactions of Influenza Fusion Proteins

Viral fusion peptides interact with lipid membranes to promote membrane fusion and viral entry. Data on fusion-defective mutants of influenza virus suggest that these interactions are in fact required for efficient fusion. Fusion peptides appear to be at least structurally plastic if not highly dynamic, adopting multiple conformations and potentially multiple interaction modes with lipid membranes. It remains unclear, however, how these structural properties are linked to physiologic function. We have used molecular dynamics simulation to probe the conformational transitions and membrane interactions of influenza hemagglutinin. Based on these simulations, we obtain models of 1) how fusion peptides can lower the activation barrier to the first lipidic intermediates in membrane fusion, 2) how structural plasticity may be important to this activity, and 3) how inactive mutants of influenza fusion peptides may be functionally impaired. We have also performed functional experiments on how the membrane environment affects fusion kinetics of live influenza virus. Correlation of models from simulation with spectroscopic and functional data can help to further elucidate the mechanisms by which fusion peptides promote membrane fusion and how structure and dynamics relate to function.

Trimeric Transmembrane Domain Interactions in Paramyxovirus Fusion Proteins: ROLES IN PROTEIN FOLDING, STABILITY, AND FUNCTION

Paramyxovirus fusion (F) proteins promote membrane fusion between the viral envelope and host cell membranes, a critical early step in viral infection. Although mutational analyses have indicated that transmembrane (TM) domain residues can affect folding or function of viral fusion proteins, direct analysis of TM-TM interactions has proved challenging. To directly assess TM interactions, the oligomeric state of purified chimeric proteins containing the Staphylococcal nuclease (SN) protein linked to the TM segments from three paramyxovirus F proteins was analyzed by sedimentation equilibrium analysis in detergent and buffer conditions that allowed density matching. A monomer-trimer equilibrium best fit was found for all three SN-TM constructs tested, and similar fits were obtained with peptides corresponding to just the TM region of two different paramyxovirus F proteins. These findings demonstrate for the first time that class I viral fusion protein TM domains can self-associate as trimeric complexes in the absence of the rest of the protein. Glycine residues have been implicated in TM helix interactions, so the effect of mutations at Hendra F Gly-508 was assessed in the context of the whole F protein. Mutations G508I or G508L resulted in decreased cell surface expression of the fusogenic form, consistent with decreased stability of the prefusion form of the protein. Sedimentation equilibrium analysis of TM domains containing these mutations gave higher relative association constants, suggesting altered TM-TM interactions. Overall, these results suggest that trimeric TM interactions are important driving forces for protein folding, stability and membrane fusion promotion.

Endocytic Rabs in membrane trafficking and signaling

The endolysosomal system controls the trafficking of proteins between the plasma membrane and the degradative environment of the lysosome. The early endosomal Rab5 and the late endosomal Rab7 GTPases have a key role in the transport along the endocytic pathway by recruiting tethering factors such as the hexameric CORVET and HOPS complexes that promote membrane fusion. Both Rabs are also involved in signaling at endosomal membranes and linked to amino acid sensing and autophagy, indicating that their role in trafficking may be connected to signal transduction and adaptation during cell stress. Here, we will summarize the current knowledge on the role of both Rab GTPases on both processes and discuss the possible crosstalk between them.

Processive ATP-driven Substrate Disassembly by the N-Ethylmaleimide-sensitive Factor (NSF) Molecular Machine

SNARE proteins promote membrane fusion by forming a four-stranded parallel helical bundle that brings the membranes into close proximity. Post-fusion, the complex is disassembled by an AAA+ ATPase called N-ethylmaleimide-sensitive factor (NSF). We present evidence that NSF uses a processive unwinding mechanism to disassemble SNARE proteins. Using a real-time disassembly assay based on fluorescence dequenching, we correlate NSF-driven disassembly rates with the SNARE-activated ATPase activity of NSF. Neuronal SNAREs activate the ATPase rate of NSF by ∼26-fold. One SNARE complex takes an average of ∼5 s to disassemble in a process that consumes ∼50 ATP. Investigations of substrate requirements show that NSF is capable of disassembling a truncated SNARE substrate consisting of only the core SNARE domain, but not an unrelated four-stranded coiled-coil. NSF can also disassemble an engineered double-length SNARE complex, suggesting a processive unwinding mechanism. We further investigated processivity using single-turnover experiments, which show that SNAREs can be unwound in a single encounter with NSF. We propose a processive helicase-like mechanism for NSF in which ∼1 residue is unwound for every hydrolyzed ATP molecule.

A Conserved Role for Atlastin GTPases in Regulating Lipid Droplet Size

Lipid droplets (LDs) are the major fat storage organelles in eukaryotic cells, but how their size is regulatedis unknown. Using genetic screens in C.elegans for LD morphology defects in intestinal cells, we found that mutations in atlastin, a GTPase required for homotypic fusion of endoplasmic reticulum (ER) membranes, cause not only ER morphology defects, but also a reduction in LD size. Similar results were obtained after depletion of atlastin or expression of a dominant-negative mutant, whereas overexpression of atlastin had the opposite effect. Atlastin depletion in Drosophila fat bodies also reduced LD size and decreased triglycerides in whole animals, sensitizing them to starvation. In mammalian cells, co-overexpression of atlastin-1 and REEP1, a paralog of the ER tubule-shaping protein DP1/REEP5, generates large LDs. The effect of atlastin-1 on LD size correlates with its activity to promote membrane fusion invitro. Our results indicate that atlastin-mediated fusion of ER membranes is important for LD size regulation.

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.

At Low pH, Influenza Virus Matrix Protein M1 Undergoes a Conformational Change Prior to Dissociating from the Membrane

The M1 matrix protein of influenza A virus, which plays multiple roles in virion assembly and infection, underlies the viral envelope. However, previous studies have given differing accounts of the number of layers in the M1-envelope complex and their thicknesses and compositions. To resolve this issue, we performed cryo-electron microscopy and cryo-electron tomography on the self-same specimens. At neutral pH, there were two kinds of complexes, corresponding to a lipid bilayer with embedded glycoproteins, with and without a closely associated, 4-nm-thick sheet of M1 protein. The reported discrepancies arose from differences in imaging conditions, i.e., in defocus and in whole-particle projections versus thin tomographic slices. Exposure of virions to low pH (as in the endosome) promotes membrane fusion, and previous work has shown that the M2 ion channel causes the virion interior to acidify also. We found that after 5 min at pH 4.9, the proportion of virions lacking an M1 layer increased from 10% to 50%. In virions retaining an M1 layer under these conditions, the M1-envelope complex exhibited two states, viz, the original, neutral-pH state and one in which the M1 layer appeared thinner and/or closer to the membrane. These observations extend previous indications that acidic pH causes the M1 layer to dissociate, leaving the envelope more pliable and, consequently, fusion compatible, and they show that dissociation is preceded by a conformational change in M1.

HIV Fusion Peptide Perturbs Membrane Structure in a Cholesterol Dependent Fashion

The fusion between viral envelopes with host cell membranes is required for viral entry and infection, which is mediated by special glycoproteins anchored on the viral membrane. Fusion peptide (FP) is the domain that initiates membrane fusion. However, the mechanism of membrane fusion is still unclear. We previously found that the influenza hemagglutinin FP increases the order of DMPC lipid bilayer. We hypothesize that inducing lipid ordering might be a critical step in fusion caused by a variety of fusion proteins. HIV gp41 FP plays a similar role as influenza HA FP. However, gp41 FP is polymorphic and changes from alpha helix to beta aggregation as cholesterol concentration in lipid increases. We used PC spin labels on the lipid head group and different positions on the acyl chain to detect the perturbation by gp41 FP to POPC/POPG lipid bilayers with different cholesterol concentration (0% to 30%) by CW-ESR. Our data show that 1) gp41 FP affects the lipid order in the same pattern as HA FP does, i.e., a cooperative effect vs. lipid/peptide ratio, thus supporting our hypothesis; 2) gp41 FP induces membrane ordering in all tested lipid compositions, consistent with promoting membrane fusion in these compositions; 3) in the high cholesterol containing lipid bilayers, whereas gp41 FP is in the beta aggregation conformation, its effect on the lipid ordering reaches deeper into the bilayer, consistent with deeper membrane insertion for gp41 FP in this conformation. We are extending the ESR studies to look for coexisting membrane microdomains induced by different conformations of FPs and the FP partitioning between them and for precise separation of the effects of ordering and molecular motion.