A Coronin 1-Derived Peptide Inhibits Membrane Fusion by Modulating Membrane Organization and Dynamics.

Membrane fusion is considered the first step in the entry of enveloped viruses into the host cell. Several targeted strategies have been implemented to block viral entry by limiting the fusion protein to form a six-helix bundle, which is a prerequisite for fusion. Nonetheless, the development of broad-spectrum fusion inhibitors is essential to combat emerging and re-emerging viral infections. TG-23, a coronin 1, a tryptophan-aspartate-rich phagosomal protein-derived peptide, demonstrated inhibition of fusion between small unilamellar vesicles (SUVs) by modulating the membrane's physical properties. However, its inhibitory efficacy reduces with an increasing concentration of membrane cholesterol. The present work aims to develop a fusion inhibitor whose efficacy would be unaltered in the presence of membrane cholesterol. A stretch of the tryptophan-aspartic acid-containing peptide with a similar secondary structure and hydrophobicity profile of TG-23 from coronin 1 was synthesized, and its ability to inhibit SUV-SUV fusion with varying concentrations of membrane cholesterol was evaluated. Our results demonstrate that the GG-21 peptide inhibits fusion irrespective of the cholesterol content of the membrane. We have further evaluated the peptide-induced change in the membrane organization and dynamics utilizing arrays of steady-state and time-resolved fluorescence measurements and correlated these results with their effect on fusion. Interestingly, GG-21 displays inhibitory efficacy in a wide variety of lipid compositions despite having a secondary structure and physical properties similar to those of TG-23. Overall, our results advocate that the secondary structure and physical properties of the peptide may not be sufficient to predict its inhibitory efficacy.

Phosphatidylglycerol Acts as a Switch for Cholesterol-Dependent Membrane Binding of ApoE Signal Peptide.

The apolipoprotein E (ApoE) signal peptide is a short stretch of N-terminal amino acids that direct the ApoE protein to the endoplasmic reticulum after synthesis. Previous studies have shown that this peptide can bind to lipid membranes in a cholesterol-dependent manner; however, the mechanism of this interaction is yet to be clarified. In this study, we aimed to investigate how the composition of neighboring lipids affects the membrane-binding of the ApoE signal peptide. We found that a negatively charged lipid, such as phosphatidylglycerol, can act as a switch that reduces the binding efficiency of the peptide to cholesterol-rich membranes. Interestingly, phosphatidylethanolamine does not activate the cholesterol-dependent binding of the ApoE signal peptide yet acts synergistically to enhance the cholesterol sensitivity in phosphatidylglycerol-containing membranes. To the best of our knowledge, this is the first report of modulation of the affinity of a peptide for a membrane by a neighboring lipid rather than by the lipid-binding domain of the peptide. Our findings revealed a novel role of lipid diversity in modulating the membrane binding of the ApoE signal peptide and its potential implications in the unidirectional trafficking of a newly synthesized protein from the ribosomes to the endoplasmic reticulum.

Peptide-Induced Fusion of Dynamic Membrane Nanodomains: Implications in a Viral Entry.

Enveloped viruses infect host cells via protein-mediated membrane fusion. However, insights into the microscopic rearrangement induced by the viral proteins and peptides have not yet emerged. Here, we report a new methodology to extract viral fusion peptide (FP)-mediated biomembrane dynamical nanodomain fusion parameter, λ, based on stimulated emission depletion microscopy coupled with fluorescence correlation spectroscopy. We also define another dynamical parameter membrane gradient, defined in terms of the ratio of average lipid diffusion coefficients across dynamic crossover length scales, ξ. Significantly, we observe that λ as well as these mobility gradients are larger in the stiffer liquid-ordered (Lo) phase compared to the liquid-disordered phase and are more effective at the smaller nanodomain interfaces, which are only present in the Lo phase. The results could possibly help to resolve a long-standing puzzle about the enhanced fusogenicity of FP in the Lo phase. Results obtained from the diffusion results have been correlated with the human immunodeficiency virus gp41 FP-induced membrane fusion.

Membrane cholesterol regulates the oligomerization and fusogenicity of SARS-CoV fusion peptide: implications in viral entry.

N-terminal residues (770-788) of the S2 glycoprotein of severe acute respiratory syndrome coronavirus (SARS-CoV) have been recognized as a potential fusion peptide that can be involved in the entry of the virus into the host cell. Membrane composition plays an important role in lipid-peptide interaction and the oligomeric status of the peptide. SARS-CoV fusion peptide (S2 fusion peptide) is known to undergo cholesterol-dependent oligomerization in the membrane; however, its significance in membrane fusion is still speculative. This study aimed to investigate the oligomerization of SARS-CoV fusion peptide in a membrane containing phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol, with varying concentrations of cholesterol, and to evaluate peptide-induced membrane fusion to correlate the importance of peptide oligomerization with membrane fusion. Peptide-induced modulation of membrane organization and dynamics was explored by steady-state and time-resolved fluorescence spectroscopic measurements using depth-dependent probes. The results clearly demonstrated the induction of S2 fusion peptide oligomerization by membrane cholesterol and the higher efficiency of the oligomer in promoting membrane fusion compared to its monomeric counterpart. Cholesterol-dependent peptide oligomerization and membrane fusion are important aspects of viral infection since the cholesterol level can change with age as well as with the onset of various pathophysiological conditions.

Combination of Oleic Acid and the gp41 Fusion Peptide Switches the Phosphatidylethanolamine-Induced Membrane Fusion Mechanism from a Nonclassical to a Classical Stalk Model.

Membrane fusion is considered to be one of the crucial processes for the existence of eukaryotes and the entry of enveloped viruses into host cells. The fusion mechanism depends on the lipid composition of the membrane as well as the properties of fusion proteins or peptides. The gp41 fusion peptide from the human immunodeficiency virus (HIV) is known to catalyze membrane fusion by altering the physical properties of the membrane. Earlier, we demonstrated that a membrane containing 30 mol % phosphatidylethanolamine (PE) circumvents the classical stalk model because of its intrinsic negative curvature. In this work, we demonstrated how the gp41 fusion peptide influences the fusion mechanism of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1,2-dioleoyl-sn-glycero-3-phos-pho¬ethanolamine (DOPE) (70/30 mol %) membranes. We further evaluated the effect of the same peptide on the mechanism of fusion for membranes containing 30 mol % PE and a fatty acid with an intrinsic positive curvature (oleic acid (OA)). Our results show that gp41 switches the fusion mechanism from a nonclassical to a classical stalk model when membranes contain OA, but fails to do so for DOPC/DOPE membranes. This could be due to the extreme influence of the intrinsic negative curvature of PE, which is partially downregulated in the presence of OA.

Mechanism of Membrane Fusion: Interplay of Lipid and Peptide.

Membrane fusion is an essential process for the survival of eukaryotes and the entry of enveloped viruses into host cells. A proper understanding of the mechanism of membrane fusion would provide us a handle to manipulate several biological pathways, and design efficient vaccines against emerging and re-emerging viral infections. Although fusion proteins take the central stage in catalyzing the process, role of lipid composition is also of paramount importance. Lipid composition modulates membrane organization and dynamics and impacts the lipid-protein (peptide) interaction. Moreover, the intrinsic curvature of lipids has strong impact on the formation of stalk and hemifusion diaphragm. Detection of transiently stable intermediates remains the bottleneck in the understanding of fusion mechanism. In order to circumvent this challenge, analytical methods can be employed to determine the kinetic parameters from ensemble average measurements of observables, such as lipid mixing, content mixing, and content leakage. The current review aims to present an analytical method that would aid our understanding of the fusion mechanism, provides a better insight into the role of lipid shape, and discusses the interplay of lipid and peptide in membrane fusion.

Trh positive strain of Vibrio parahaemolyticus induce immunity by modulating MAPK pathway: A molecular pathogenic insight in immune-related gene regulation.

Vibrio parahaemolyticus is a zoonotic bacterium that causes infections in shellfish, fish and higher vertebrates as well as in humans. The Tdh and Trh positive strains of V. parahaemolyticus are generally considered as major virulent strains. The pathogenic mechanisms of Trh positive strain of V. parahaemolyticus are poorly understood. Therefore, in the present study Indian Major Carp, Labeo rohita was intraperitoneally challenged with a Trh positive strain of V. parahaemolyticus below lethal dose 50 (LD50) to understand the innate immune response. A significant upregulation in the respiratory burst activity, myeloperoxidase activity and lysozyme activity of serum was observed in the challenged fishes. However, the serum alpha (α) 2-macro globulin activity and antiprotease activity remained unaltered in the infected fish. The relative expression study of some immune-related genes showed that after the experimental challenge the expression of immune-related genes viz., Toll-like receptor (TLR), Nucleotide-binding oligomerization domain (NOD), Interleukin-1ß (IL-ß), Interleukin-6 (IL-6), Tumor necrosis factor α (TNFα), Inducible nitric oxide synthase (iNOS), Complement factor 3a (C3a) and Heat shock proteins 70 (Hsp70) was upregulated during infection. Furthermore, overexpression of nuclear factor kappa light chain enhancer of activated B cells (NF-κß), Myeloid differentiation primary response 88 (MyD88), Mitogen-activated protein kinases (MAPK) and cysteine-aspartic proteases (Casp 1) was also observed after post-infection which clearly indicated that Trh positive V. parahaemolyticus activates MAPK pathway. The present study strengthens the understanding of molecular pathogenesis and provides insights on gene regulation during infection with Trh positive V. parahaemolyticus.

Cholesterol: A key player in membrane fusion that modulates the efficacy of fusion inhibitor peptides.

The interaction of cholesterol with the neighboring lipids modulates several physical properties of the membrane. Mostly, it affects membrane fluidity, membrane permeability, lateral diffusion of lipids, bilayer thickness, and water penetration into the lipid bilayer. Due to the smaller head group to hydrophobic cross-sectional area of the tail, cholesterol induces intrinsic negative curvature to the membrane. The interaction of cholesterol with sphingolipids forms lipid rafts; generates phase separation in the membrane. The cholesterol-dependent modifications of membrane physical properties modulate viral infections by affecting the fusion between viral and host cell membranes. Cholesterol demonstrates a strong impact on the structure, depth of penetration, conformation, and organization of fusion peptides in membrane milieu. Further, cholesterol has been implicated to modify the fusion inhibitory efficiency of peptide-based membrane fusion inhibitors.

The role of fusion peptides in depth-dependent membrane organization and dynamics in promoting membrane fusion.

Membrane fusion is an important event in the life of eukaryotes; occurs in several processes such as endocytosis, exocytosis, cellular trafficking, compartmentalization, import of nutrients and export of waste, vesiculation, inter cellular communication, and fertilization. The enveloped viruses as well utilize fusion between the viral envelope and host cell membrane for infection. The stretch of 20-25 amino acids located at the N-terminus of the fusion protein, known as fusion peptide, plays a decisive role in the fusion process. The stalk model of membrane fusion postulated a common route of bilayer transformation for stalk, transmembrane contact, and pore formation; and fusion peptide is believed to facilitate bilayer transformation to promote membrane fusion. The peptide-induced change in depth-dependent organization and dynamics could provide important information in understanding the role of fusion peptide in membrane fusion. In this review, we have discussed about three depth-dependent properties of the membrane such as rigidity, polarity and heterogeneity, and the impact of fusion peptide on these three membrane properties.

Mechanistic insights of host cell fusion of SARS-CoV-1 and SARS-CoV-2 from atomic resolution structure and membrane dynamics.

The emerging and re-emerging viral diseases are continuous threats to the wellbeing of human life. Previous outbreaks of Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS had evidenced potential threats of coronaviruses in human health. The recent pandemic due to SARS-CoV-2 is overwhelming and has been going beyond control. Vaccines and antiviral drugs are ungently required to mitigate the pandemic. Therefore, it is important to comprehend the mechanistic details of viral infection process. The fusion between host cell and virus being the first step of infection, understanding the fusion mechanism could provide crucial information to intervene the infection process. Interestingly, all enveloped viruses contain fusion protein on their envelope that acts as fusion machine. For coronaviruses, the spike or S glycoprotein mediates successful infection through receptor binding and cell fusion. The cell fusion process requires merging of virus and host cell membranes, and that is essentially performed by the S2 domain of the S glycoprotein. In this review, we have discussed cell fusion mechanism of SARS-CoV-1 from available atomic resolution structures and membrane binding of fusion peptides. We have further discussed about the cell fusion of SARS-CoV-2 in the context of present pandemic situation.

Differential sensitivity of pHLIP to ester and ether lipids.

pH (low) insertion peptide (pHLIP) is a polypeptide from the third transmembrane helix of bacteriorhodopsin. The pH-dependent membrane insertion of pHLIP has been conveniently exploited for translocation of cargo molecules and as a novel imaging agent in cancer biology due to low extracellular pH in cancer tissues. Although the application of pHLIP for imaging tumor and targeted drug delivery is well studied, literature on pHLIP-membrane interaction is relatively less studied. Keeping this in mind, we explored the differential interaction of pHLIP with ester and ether lipid membranes utilizing fluorescence and CD spectroscopy. We report, for the first time, higher binding affinity of pHLIP toward ether lipid relative to ester lipid membranes. There results gain relevance since Halobacterium halobium (source of bacteriorhodopsin) is enriched with ether lipids. In addition, we monitored the difference in microenvironment around pHLIP tryptophans utilizing red edge excitation shift and observed increased motional restriction of water molecules in the interfacial region in ether lipid membranes. These changes were accompanied with increase in helicity of pHLIP in ether lipid relative to ester lipid membranes. Our results assume further relevance since ether lipids are upregulated in cancer cells and have emerged as potential biomarkers of various diseases including cancer.

Membrane Cholesterol Modulates Oligomeric Status and Peptide-Membrane Interaction of Severe Acute Respiratory Syndrome Coronavirus Fusion Peptide.

The N-terminal fusion peptide (residues 770-788) of an S2 glycoprotein of the severe acute respiratory syndrome coronavirus (SARS-CoV), exposed upon receptor binding, is crucial for virus entry into the host cell. The fusion peptide alters the membrane organization and dynamics of the host membrane to facilitate membrane fusion. Generally, the effect of the fusion peptide on the membrane is sensitive to the lipid composition of target membranes. In the present work, we have utilized steady-state and time-resolved fluorescence spectroscopy in tandem with circular dichroism spectroscopy to elucidate the binding, oligomeric status, and secondary structure of the fusion peptide and its impact on the depth-dependent membrane organization and dynamics. We have used depth-dependent fluorescence probes, 1,6-diphenyl-1,3,5-hexatriene (DPH) and its trimethylammonium derivative (TMA-DPH), to evaluate the effect of the peptide binding along the bilayer normal. We have exploited the energy transfer efficiency of tryptophan between TMA-DPH and DPH to determine the relative location of the solitary tryptophan present in the membrane-bound fusion peptide. We have further evaluated the effect of membrane cholesterol on the binding and organization of the peptide and the impact of peptide binding on the depth-dependent physical properties of the membrane at various cholesterol concentrations. Our results clearly demonstrate that the membrane cholesterol alters the oligomeric status of the membrane-bound peptide and the effect of peptide binding on the depth-dependent membrane organization and dynamics. The role of cholesterol is important, as the eukaryotic host cells contain a good amount of cholesterol that might be important for the entry of pathogenic viruses.

Cholesterol alters the inhibitory efficiency of peptide-based membrane fusion inhibitor.

The membrane composition modulates membrane fusion by altering membrane physical properties and the structure, organization and dynamics of fusion proteins and peptides. The journey of developing peptide-based viral fusion inhibitors is often stalled by the change in lipid composition of viral and target membranes. This makes it important to study the role of membrane composition on the organization, dynamics and fusion inhibiting abilities of the peptide-based fusion inhibitors. Cholesterol, an important constituent of mammalian cell membrane, modulates bilayer properties in multiple ways and impart its effect on the membrane fusion. We have previously shown that TG-23 peptide derived from phagosomal coat protein, coronin 1, shows significant inhibition of fusion between membranes without cholesterol. In this work, we have studied the effect of the TG-23 peptide on the polyethylene glycol-mediated membrane fusion in presence of different concentrations of membrane cholesterol. Our results show that the inhibitory effect of TG-23 is being completely reversed in cholesterol containing membranes. We have evaluated the structure, organization, dynamics and depth of penetration of TG-23 in membranes having different lipid compositions and its effect on membrane properties. Our results demonstrate that cholesterol does not affect the secondary structure of the peptide, however, alters the depth of penetration of the peptide and modifies peptide organization and dynamics. The cholesterol dependent change in organization and dynamics of the peptide influences its efficacy in membrane fusion. Therefore, we envisage that the study of peptide organization and dynamics is extremely important to determine the effect of peptide on the membrane fusion.

Membrane Composition Modulates Fusion by Altering Membrane Properties and Fusion Peptide Structure.

Membrane fusion, one of the most essential processes in the life of eukaryotes, occurs when two separate lipid bilayers merge into a continuous bilayer and internal contents of two separated membranes mingle. There is a certain class of proteins that assist the binding of the viral envelope to the target host cell and catalyzing fusion. All class I viral fusion proteins contain a highly conserved 20-25 amino-acid amphipathic peptide at the N-terminus, which is essential for fusion activity and is termed as the 'fusion peptide'. It has been shown that insertion of fusion peptides into the host membrane and the perturbation in the membrane generated thereby is crucial for membrane fusion. Significant efforts have been given in the last couple of decades to understand the lipid-dependence of structure and function of the fusion peptide in membranes to understand the role of lipid compositions in membrane fusion. In addition, the lipid compositions further change the membrane physical properties and alter the mechanism and extent of membrane fusion. Therefore, lipid compositions modulate membrane fusion by changing membrane physical properties and altering structure of the fusion peptide.

Therapeutic value of steroidal alkaloids in cancer: Current trends and future perspectives.

Discovery and development of new potentially selective anticancer agents are necessary to prevent a global cancer health crisis. Currently, alternative medicinal agents derived from plants have been extensively investigated to develop anticancer drugs with fewer adverse effects. Among them, steroidal alkaloids are conventional secondary metabolites that comprise an important class of natural products found in plants, marine organisms and invertebrates, and constitute a judicious choice as potential anti-cancer leads. Traditional medicine and modern science have shown that representatives from this compound group possess potential antimicrobial, analgesic, anticancer and anti-inflammatory effects. Therefore, systematic and recapitulated information about the bioactivity of these compounds, with special emphasis on the molecular or cellular mechanisms, is of high interest. In this review, we methodically discuss the in vitro and in vivo potential of the anticancer activity of natural steroidal alkaloids and their synthetic and semi-synthetic derivatives. This review focuses on cumulative and comprehensive molecular mechanisms, which will help researchers understand the molecular pathways involving steroid alkaloids to generate a selective and safe new lead compound with improved therapeutic applications for cancer prevention and therapy. In vitro and in vivo studies provide evidence about the promising therapeutic potential of steroidal alkaloids in various cancer cell lines, but advanced pharmacokinetic and clinical experiments are required to develop more selective and safe drugs for cancer treatment.

Coronin 1 derived tryptophan-aspartic acid containing peptides inhibit membrane fusion.

Membrane fusion is an integral part of the viral infection. The fusion between an enveloped virus and a host cell is the first step for viral infection. It has been a long-standing effort to develop anti-viral therapies involving inhibitors that block the fusion between virus and host cell. However, these inhibitors are highly specific against a particular virus. Development of generic inhibitors is extremely essential in the current scenario to overcome emerging and re-emerging contagious diseases that cause millions of fatalities every year. In this work, we have studied the effect of three different peptides derived from a phagosomal protein coronin 1. Coronin 1 is being recruited at the phagosomal membrane of Mycobacterium infected host cells and is implicated in preventing lysosomal fusion. Interestingly, coronin 1 contains tryptophan-aspartic acid repeats, which are conserved across species. In order to understand the mechanistic basis of coronin 1 function, we designed peptides that contain conserved tryptophan-aspartic acid region, and evaluated their membrane binding, effect on membrane fusion, depth-dependent membrane ordering and water penetration into the membrane. Our results demonstrate that these peptides exclusively bind to membranes in presence of negatively charged lipids and do not influence lipid mixing. However, two peptides, TG-23 and GL-22, substantially reduce the extent of content mixing. The reduction in content mixing in presence of TG-23 and GL-22 could be interpreted in terms of their inhibitory effect on water penetration into the membrane. We envisage that these results will contribute to the development of the generic peptide-based membrane fusion inhibitors.

Aggregation Behavior of pHLIP in Aqueous Solution at Low Concentrations: A Fluorescence Study.

pH (low) insertion peptide (pHLIP) is a 36-residue peptide derived from the third transmembrane helix of the membrane protein bacteriorhodopsin. The hydrophobicity of this peptide makes it prone to aggregation even at low concentrations, but this has not been studied in detail. In this work, we characterized monomeric and aggregated forms of pHLIP in aqueous solution (pH 8) at low concentrations (~µM) using fluorescence-based approaches, complemented by circular dichroism (CD) spectroscopy. We show here that monomeric and aggregated pHLIP display differential red edge excitation shift (REES) and CD spectra. These spectroscopic features allowed us to show that pHLIP aggregates even at low concentrations. A detailed knowledge of the aggregation behavior of pHLIP under these conditions will be useful for monitoring and quantifying its interaction with membranes.

Exploring the Mechanism of Viral Peptide-Induced Membrane Fusion.

Membrane fusion is essential in several cellular processes in the existence of eukaryotic cells such as cellular trafficking, compartmentalization, intercellular communication, sexual reproduction, cell division, and endo- and exocytosis. Membrane fusion proceeds in model membranes as well as biological membranes through the rearrangement of lipids. The stalk hypothesis provides a picture of the general nature of lipid rearrangement based on mechanical properties and phase behavior of water-lipid mesomorphic systems. In spite of extensive research on exploring the mechanism of membrane fusion, a clear molecular understanding of intermediate and pore formation is lacking. In addition, the mechanism by which proteins and peptides reduce the activation energy for stalk and pore formation is not yet clear though there are several propositions on how they catalyze membrane fusion. In this review, we have discussed about various putative functions of fusion peptides by which they reduce activation barrier and thus promote membrane fusion. A careful analysis of the discussed effects of fusion peptides on membranes might open up new possibilities for better understanding of the membrane fusion mechanism.

Targeting the dengue ß-OG with serotype-specific alkaloid virtual leads.

The dengue envelope ß-OG pocket is a crucial hinge for mediating virus-host fusion via conformational changes in the envelope to the fusion-competent form. The ß-OG pocket is a small molecule target site for inhibition of virus-host fusion. As of date, the only structure of the ß-OG pocket known is of serotype 2. Studies of ß-OG inhibition by small molecules primarily target viral serotype 2. Envelope and ß-OG sequence alignments, reveal dissimilarities across serotypes. In light of protein sequence-structure-function correlation, sequence variations suggest serotypic variations in ß-OG druggability. This, together with the fact that dengue viral proteins do have serotype-specific variations of structure and function, lead to the study of the serotype-specificity of the dengue ß-OG ligand binding behaviour. ß-OG druggability was compared using comparative models of envelope proteins containing the ß-OG pocket in four serotypes of the dengue virus. ß-OG ligand binding was found to vary with respect to hydrophobicity, hydrophilicity, hydrogen bonding, van der Waals interactions with ligands and tightness of the binding site. The study also reports serotype-specific virtual leads identified from a library of 9175 alkaloids, using a consensus docking and scoring approach. The docking algorithms of Glide SP and XP, together with the Lamarckian genetic algorithm were employed for consensus docking. For consensus scoring, the Glide empirical score was employed along with the scoring function of AutoDock. A multi-dimensional lead optimisation approach was performed for optimising affinity, ligand efficiency, lipophilic ligand efficiency, ADMET and molecular torsional strains. The study proposes the serotype-specific inhibition of the ß-OG for an effective inhibition of virus-host fusion, in contrast to a pan inhibitor.

Depth-Dependent Membrane Ordering by Hemagglutinin Fusion Peptide Promotes Fusion.

Membrane fusion, one of the most fundamental processes in life, occurs when two separate lipid membranes merge into a single continuous bilayer. Membrane fusion is essential for the entry of lipid-sheathed viruses such as influenza and HIV. Influenza virus is internalized via receptor-mediated endocytosis and then fuses with the endosomal membrane at low pH. Hemagglutinin, a glycoprotein found on the surface of influenza virus, is responsible for the fusion of the viral sheath with the endosomal membrane. The ∼20 amino acid long N-terminus of hemagglutinin, known as the fusion peptide, plays a crucial role in the viral fusion process. Although there exists vast literature on the importance and role of the fusion peptide in promoting membrane fusion, there is no consensus on the mechanism by which it promotes fusion. A recent report suggested that the fusion peptide occupies and orders space in the outer leaflets of contacting bilayers so as to promote acyl chain protrusion into interbilayer space and promote fusion "stalk" formation. We report here the effect of the wild type, G1S, G1V, and W14A mutants of hemagglutinin fusion peptide on depth-dependent ordering of model membranes along the bilayer normal. We utilized fluorescence anisotropy, lifetime measurements, and lifetime distribution analyses of different anthroyloxy stearic acid probes (n-AS) in order to examine the effect of fusion peptides at various depths along the bilayer normal. Wild type peptide uniquely ordered a region ∼12 Šfrom the bilayer midpoint, W14A and G1S mutants mainly ordered the bilayer interface, while G1V had little ordering influence. On the basis of recent analysis of the effects of these peptides on fusion, ordering of the mid-upper region of the bilayer appears to promote fusion pore formation, while ordering of the bilayer interface inhibits it.