Binding of glucose to the D-galactose/D-glucose-binding protein from Escherichia coli restores the native protein secondary structure and thermostability that are lost upon calcium depletion

The effect of the depletion of calcium on the structure and thermal stability of the D-galactose/D-glucose-binding protein (GGBP) from Escherichia coli was studied by fluorescence spectroscopy and Fourier-transform infrared spectroscopy. The calcium-depleted protein (GGBP-Ca) was also studied in the presence of glucose (GGBP-Ca/Glc). The results show that calcium depletion has a small effect on the secondary structure of GGBP, and, in particular it affects a population of alpha-helices with a low exposure to solvent. Alternatively, glucose-binding to GGBP-Ca eliminates the effect induced by calcium depletion by restoring a secondary structure similar to that of the native protein. In addition, the infrared and fluorescence data obtained reveal that calcium depletion markedly reduces the thermal stability of GGBP. In particular, the spectroscopic experiments show that the depletion of calcium mainly affects the stability of the C-terminal domain of the protein. However, the binding of glucose restores the thermal stability of GGBP-Ca. The thermostability of GGBP and GGBP-Ca was also studied by molecular dynamics simulations. The simulation data support the spectroscopic results. New insights into the role of calcium in the thermal stability of GGBP contribute to a better understanding of the protein function and constitute important information for the development of biotechnological applications of this protein. Mutations and/or labelling of amino acid residues located in the protein C-terminal domain may affect the stability of the whole protein structure.     

Pressure effect on the stability and the conformational dynamics of the D-Galactose/D-Glucose-binding protein from Escherichia coli

The effect of the pressure on the structure and stability of the D-Galactose/D-Glucose binding protein (GGBP) from Escherichia coli was studied by steady-state and time-resolved fluorescence spectroscopy, and the ability of glucose ligand to stabilize the GGBP structure was also investigated. Steady-state fluorescence experiments showed a marked quenching of fluorescence emission of GGBP in the absence of glucose. Instead, the presence of glucose seems to stabilize the structure of GGBP at low and moderate pressure values. Time-resolved fluorescence measurements showed that the GGBP taumean in the absence of glucose varies significantly up to 600 bar, while in the presence of the ligand it is almost unaffected by pressure increase up to 600 bar. The effect of the pressure on GGBP was also studied by molecular dynamics simulations. The simulation data support the spectroscopic results and confirm that the presence of glucose is able to contrast the negative effects of pressure on the protein structure. Taken together, the spectroscopic and computer simulation studies suggest that at pressure values up to 2000 bar the structure of GGBP in the absence of glucose remains folded, but a significant perturbation of the protein secondary structures can be detected. The binding of glucose reduces the negative effect of pressure on protein structure and confers protection from perturbation especially at moderate pressure values.     

Temperature-, SDS-, and pH-induced conformational changes in protein disulfide oxidoreductase from the archaeon Pyrococcus furiosus: a dynamic simulation and fourier transform infrared spectroscopic study

The effect of SDS, pD, and temperature on the structure and stability of the protein disulfide oxidoreductase from Pyrococcus furiosus (PfPDO) was investigated by molecular dynamic (MD) simulations and FT-IR spectroscopy. pD affects the thermostability of alpha-helices and beta-sheets differently, and 0.5% or higher SDS concentration influences the structure significantly. The experiments allowed us to detect a secondary structural reorganization at a definite temperature and pD which may correlate with a high ATPase activity of the protein. The MD simulations supported the infrared data and revealed the different behavior of the N and C terminal segments, as well as of the two active sites.     

A strategic fluorescence labeling of D-galactose/D-glucose-binding protein from Escherichia coli helps to shed light on the protein structural stability and dynamics

The D-glucose/D-galactose-binding protein (GGBP) of Escherichia coli serves as an initial component for both chemotaxis toward D-galactose and D-glucose and high-affinity active transport of the two sugars. GGBP is a monomer with a molecular weight of about 32 kDa that binds glucose with micromolar affinity. The sugar-binding site is located in the cleft between the two lobes of the bilobate protein. In this work, the local and global structural features of GGBP were investigated by a strategic fluorescence labeling procedure and spectroscopic methodologies. A mutant form of GGBP containing the amino acid substitution Met to Cys at position 182 was realized and fluorescently labeled to probe the effect of glucose binding on the local and overall structural organization of the protein. The labeling of the N-terminus with a fluorescence probe as well as the protein intrinsic fluorescence were also used to obtain a complete picture of the GGBP structure and dynamics. Our results showed that the binding of glucose to GGBP resulted in no stabilizing effect on the N-terminus portion of GGBP and in a moderate stabilization of the protein matrix in the vicinity of the ligand-binding site. On the contrary, it was observed that the binding of glucose has a strong stabilization effect on the C-terminal domain of the GGBP structure.     

Conformation and aggregation of M13 coat protein studied by molecular dynamics.

Molecular dynamics (MD) simulations are performed on M13 coat protein, a small membrane protein for which both alpha- and beta-structures have been suggested. The simulations are started from initial conformations that are either monomers or dimers of alpha-helices or U-shaped beta-sheets. The lipid bilayer is represented by a hydrophobic potential. The results are analyzed in terms of stability, energy and secondary structure. The U-shaped beta-structure changes from a planar to a twisted form with larger twist for the monomer than the dimer. The beta-sheet is much more flexible than the alpha-helix as monitored by the root mean square (rms) fluctuations of the C alpha atoms. A comparison of the energies after 100 ps MD simulation shows that of the monomers, the alpha-helix has the lowest energy. The energy difference between alpha- and beta-structures decreases from 266 kJ/mol to 148 kJ/mol, when going from monomers to dimers. It is expected that this difference will decrease with higher aggregation numbers.     

Interactions of chicken liver basic fatty acid-binding protein with lipid membranes

The interactions of chicken liver basic fatty acid-binding protein (Lb-FABP) with large unilamellar vesicles (LUVs) of palmitoyloleoyl phosphatidylcholine (POPC) and palmitoyloleoyl phosphatidylglycerol (POPG) were studied by binding assays, Fourier transform infrared (FT-IR) spectroscopy, monolayers at air-water interface, and low-angle X-ray diffraction. Lb-FABP binds to POPG LUVs at low ionic strength but not at 0.1 M NaCl. The infrared (IR) spectra of the POPG membrane-bound protein showed a decrease of the band corresponding to beta-structures as compared to the protein in solution. In addition, a cooperative decrease of the beta-edge band above 70 degrees C in solution was also evident, while the transition was less cooperative and took place at lower temperature for the POPG membrane-bound protein. Low- and wide-angle X-ray diffraction experiments with lipid multilayers indicate that binding of the protein produces a rearrangement of the membrane structure, increasing the interlamellar spacing and decreasing the compactness of the lipids.     

Engineering of ligand specificity of periplasmic binding protein for glucose sensing

A novel glucose-sensing molecule was created based on galactose/glucose-binding protein (GGBP). GGBP mutants at Asp14, a residue interacting with the 4th hydroxyl group of the sugar molecule, were constructed by mutagenesis to improve the ligand specificity of GGBP. The autofluorescence-based analysis of the binding abilities of these engineered GGBPs showed that the GGBP mutants Asp14Asn and Asp14Glu bound only to glucose in a concentration-dependent manner, without being affected by the presence of galactose. The Phe16Ala mutation, which leads to an increase in the K (d) value toward glucose, was then introduced into these two glucose-specific mutant GGBPs. One of the constructed GGBP double-mutants, Asp14Glu/Phe16Ala, had a glucose specificity with a K(d) value of 3.9 mM, which makes it suitable for use in the measurement of the physiological glucose concentration. Our results demonstrate that it is possible to construct a GGBP which specifically recognizes glucose and has a higher K(d) value and use it as a molecular recognition element of blood glucose monitoring systems by combining two different mutations based on the 3D structure of GGBP.     

Tryptophan phosphorescence studies of the D-galactose/D-glucose-binding protein from Escherichia coli provide a molecular portrait with structural and dynamics features of the protein

The D-galactose/D-glucose-binding protein (GGBP) from E. coli serves as an initial component for both chemotaxis toward glucose and high-affinity active transport of the sugar. In this work, we have used phosphorescence spectroscopy to investigate the effects of glucose and calcium on the dynamics and stability of GGBP. We found that GGBP exhibits a phosphorescence spectrum composed of two energetically distinct 0,0-vibrational bands centered at 404.43 and 409.61 nm; the large energy separation between them indicates two classes of chromophores making distinct dipolar interactions with their surrounding. Interestingly, the high-energy spectral component (404.43 nm) is one of the bluest spectra reported to date in proteins. Considering the ground state dipole direction, low-energy configurations for the indole side chain in proteins leading to blue-shifted spectra can arise from negative charges in proximity to the imidazole-ring nitrogen and/or positive charges near C4-C5 of the benzene ring. Among the five tryptophan residues of GGBP, Trp-284, located at the N-terminal domain of the protein, and Trp-183, located in the protein hinge region, make strong attractive charge interactions with surrounding side chains. Regarding Trp-284, the indole ring nitrogen is in contact with the negative charge of the Asp-267, whereas Trp-183 is next to the Glu-149 residue. In the latter, the ground state energy is further lowered by the proximity of the Arg-158 to the negative end (near C6) of the indole dipole. Regarding the red spectral component (409.61 nm), it is more intense than the blue component, presumably because more residues contribute to it. lambda 0,0 is typical of environments that are weakly polar or characterized by charges positioned near 90 degrees from the ground state dipole direction (the case of W195 and W127). The binding of glucose modifies the phosphorescence lifetime values as well as the spectrum of GGBP, shifting the blue band 0.54 nm to the blue and the red band 1 nm to the red. Finally, the removal of the calcium from GGBP structure causes variations in lifetime values and spectral shifts similar to those induced by glucose binding to the native protein. Aided by a detailed inspection of the three-dimensional structure of GGBP, these results contribute to a better understanding of the structure/function relationship of this protein.     

Structural and thermal stability characterization of Escherichia coli D-galactose/D-glucose-binding protein

The effect of temperature and glucose binding on the structure of the galactose/glucose-binding protein from Escherichia coli was investigated by circular dichroism, Fourier transform infrared spectroscopy, and steady-state and time-resolved fluorescence. The data showed that the glucose binding induces a moderate change of the secondary structure content of the protein and increases the protein thermal stability. The infrared spectroscopy data showed that some protein stretches, involved in alpha-helices and beta strand conformations, are particularly sensitive to temperature. The fluorescence studies showed that the intrinsic tryptophanyl fluorescence of the protein is well represented by a three-exponential model and that in the presence of glucose the protein adopts a structure less accessible to the solvent. The new insights on the structural properties of the galactose/glucose-binding protein can contribute to a better understanding of the protein functions and represent fundamental information for the development of biotechnological applications of the protein.     

D-trehalose/D-maltose-binding protein from the hyperthermophilic archaeon Thermococcus litoralis: the binding of trehalose and maltose results in different protein conformational states

In this work, we used fluorescence spectroscopy, molecular dynamics simulation, and Fourier transform infrared spectroscopy for investigating the effect of trehalose binding and maltose binding on the structural properties and the physical parameters of the recombinant D-trehalose/D-maltose binding protein (TMBP) from the hyperthermophilic archaeon Thermococcus litoralis. The binding of the two sugars to TMBP was studied in the temperature range 20 degrees-100 degrees C. The results show that TMBP possesses remarkable temperature stability and its secondary structure does not melt up to 90 degrees C. Although both the secondary structure itself and the sequence of melting events were not significantly affected by the sugar binding, the protein assumes different conformations with different physical properties depending whether maltose or trehalose is bound to the protein. At low and moderate temperatures, TMBP possesses a structure that is highly compact both in the absence and in the presence of two sugars. At about 90 degrees C, the structure of the unliganded TMBP partially relaxes whereas both the TMBP/maltose and the TMBP/trehalose complexes remain in the compact state. In addition, Fourier transform infrared results show that the population of alpha-helices exposed to the solvent was smaller in the absence than in the presence of the two sugars. The spectroscopic results are supported by molecular dynamics simulations. Our data on dynamics and stability of TMBP can contribute to a better understanding of transport-related functions of TMBP and constitute ground for targeted modifications of this protein for potential biotechnological applications.     

A thermostable sugar-binding protein from the Archaeon Pyrococcus horikoshii as a probe for the development of a stable fluorescence biosensor for diabetic patients

In this work is presented the first attempt to develop an innovative ultrastable protein-based biosensor for blood glucose detections. The gene of a putative thermostable sugar-binding protein has been cloned and expressed in E. coli. The recombinant protein has been purified to homogeneity by thermoprecipitation and affinity chromatography steps. The recombinant protein is a monomer with an apparent molecular weight of 55,000 as judged by gel filtration and sodium dodecyl sulfate polyacrylamide gel eletrophoresis. Circular dichroism experiments showed that the protein possesses a secondary structure content rich in alpha-helices and beta-structures and that the protein is highly stable as investigated in the range of temperature between 20 and 95 degrees C. Fluorescence spectroscopy experiments demonstrated that the recombinant protein binds glucose with a dissociation constant of about 10 mM, a concentration of sugar very close to the concentration of glucose present in the human blood. A docking simulation on the modeled structure of the protein confirms its ability to bind glucose and proposes possible modifications to improve the affinity for glucose and/or its detection. The obtained results suggest the use of the protein as a probe for a stable glucose biosensor.     

Comparison of the periplasmic receptors for L-arabinose, D-glucose/D-galactose, and D-ribose. Structural and Functional Similarity.

The primary sequence of the receptor for L-arabinose or Ara-binding protein (ABP) composed of 306 residues is very different from the D-glucose/D-galactose-binding protein (GGBP) which consists of 309 residues. Nevertheless, superimpositioning of the well-refined high resolution structures of ABP in complex with D-galactose and the GGBP in complex with D-glucose shows very similar structures; 220 of the residues (or about 70%) have a root mean square deviation of 2.0 A. From the superpositioning, nine pairs of continuous segments (consisting of 8-51 residues), mainly alpha-helices and beta-strands that form the core of the two lobes of the bilobate proteins were found to exhibit strong sequence homology. The equivalenced structures and aligned sequences show that many of the polar, as well as aromatic residues, in the sugar-binding sites located in the cleft between the two lobes are highly conserved. Surprisingly, however, the exact mode of binding of the D-galactose in ABP is totally different from that of the D-glucose in GGBP. Using the structurally aligned sequences of the ABP and GGBP as a template, we have matched the sequence of the ribose-binding protein (RBP) which consists of 271 residues with the ABP/GGBP pair. Although the nine aligned segments of all three proteins show little sequence identity, they have significant homology. Four additional segments of RBP were matched only with GGBP, leading to the alignment of about 90% of the RBP sequence with the GGBP sequence. Many of the conserved residues in the binding sites of ABP and GGBP matched with similar residues in RBP. Additional observations indicate that the GGBP/RBP pair is more closely related than the ABP/RBP or ABP/GGBP pair. All three binding proteins, which may have diverged from a common ancestor, serve as primary receptors for bacterial high affinity active transport systems. Moreover, GGBP and RBP, but not ABP, also act as receptors for chemotaxis. An exposed site located in one domain, which includes Gly74, for interacting with the trg transmembrane signal transducer that is involved in triggering chemotaxis has been located in the structure of GGBP (Vyas, N.K., Vyas, M.N., and Quiocho, F.A. (1988) Science 242, 1290-1295). Whereas the site is absent in the structure of ABP, it is strongly predicted to be present in RBP which shares the same trg transducer with GGBP. The knowledge-based alignment of RBP further revealed two possible additional peripheral chemotactic sites that show high structural and sequence similarity between GGBP and RBP only. At least one of these sites, together with the one proven to exist in the other domain, could be used by the signal transducer with which both binding proteins interact in a way which the substrate-loaded "closed cleft" structure could be discriminated from the unliganded "open cleft" form by the transducer.     

Protein free energy landscapes remodeled by ligand binding

Glucose/galactose binding protein (GGBP) functions in two different larger systems of proteins used by enteric bacteria for molecular recognition and signaling. Here we report on the thermodynamics of conformational equilibrium distributions of GGBP. Three fluorescence components appear at zero glucose concentration and systematically transition to three components at high glucose concentration. Fluorescence anisotropy correlations, fluorescent lifetimes, thermodynamics, computational structure minimization, and literature work were used to assign the three components as open, closed, and twisted conformations of the protein. The existence of three states at all glucose concentrations indicates that the protein continuously fluctuates about its conformational state space via thermally driven state transitions; glucose biases the populations by reorganizing the free energy profile. These results and their implications are discussed in terms of the two types of specific and nonspecific interactions GGBP has with cytoplasmic membrane proteins.     

Binding and release of cholesterol in the Osh4 protein of yeast

Sterols have been shown experimentally to bind to the Osh4 protein (a homolog of the oxysterol binding proteins) of Saccharomyces cerevisiae within a binding tunnel, which consists of antiparallel beta-sheets that resemble a beta-barrel and three alpha-helices of the N-terminus. This and other Osh proteins are essential for intracellular transport of sterols and ultimately cell life. Molecular dynamics (MD) simulations are used to study the binding of cholesterol to Osh4 at the atomic level. The structure of the protein is stable during the course of all MD simulations and has little deviation from the experimental crystal structure. The conformational stability of cholesterol within the binding tunnel is aided in part by direct or water-mediated interactions between the 3-hydroxyl (3-OH) group of cholesterol and Trp(46), Gln(96), Tyr(97), Asn(165), and/or Gln(181) as well as dispersive interactions with Phe(42), Leu(24), Leu(39), Ile(167), and Ile(203). These residues along with other nonpolar residues in the binding tunnel and lid contribute nearly 75% to the total binding energy. The strongest and most populated interaction is between Gln(96) and 3-OH with a cholesterol/Gln(96) interaction energy of -4.5 +/- 1.0 kcal/mol. Phe(42) has a similar level of attraction to cholesterol with -4.1 +/- 0.3 kcal/mol. A MD simulation without the N-terminus lid that covers the binding tunnel resulted in similar binding conformations and binding energies when compared with simulations with the full-length protein. Steered MD was used to determine details of the mechanism used by Osh4 to release cholesterol to the cytoplasm. Phe(42), Gln(96), Asn(165), Gln(181), Pro(211), and Ile(206) are found to direct the cholesterol as it exits the binding tunnel as well as Lys(109). The mechanism of sterol release is conceptualized as a molecular ladder with the rungs being amino acids or water-mediated amino acids that interact with 3-OH.     

Direct detection of glucose by surface plasmon resonance with bacterial glucose/galactose-binding protein

The monitoring and management of blood glucose levels are key components for maintaining the health of people with diabetes. Traditionally, glucose monitoring has been based on indirect detection using electrochemistry and enzymes such as glucose oxidase or glucose dehydrogenase. Here, we demonstrate direct detection of glucose using a surface plasmon resonance (SPR) biosensor. By site-specifically and covalently attaching a known receptor for glucose, the glucose/galactose-binding protein (GGBP), to the SPR surface, we were able to detect glucose binding and determine equilibrium binding constants. The site-specific coupling was accomplished by mutation of single amino acids on GGBP to cysteine and subsequent thiol conjugation. The resulting SPR surfaces had glucose-specific binding properties consistent with known properties of GGBP. Further modifications were introduced to weaken GGBP-binding affinity to more closely match physiologically relevant glucose concentrations (1-30 mM). One protein with a response close to this glucose range was identified, the GGBP triple mutant E149C, A213S, L238S with an equilibrium dissociation constant of 0.5mM. These results suggest that biosensors for direct glucose detection based on SPR or similar refractive detection methods, if miniaturized, have the potential for development as continuous glucose monitoring devices.     

Polarized ATR-FTIR spectroscopy of the membrane-embedded domains of the particulate methane monooxygenase

The particulate methane monooxygenase (pMMO) of Methylococcus capsulatus (Bath) is an integral membrane protein that catalyzes the conversion of methane to methanol. To gain some insight into the structure-reactivity pattern of this protein, we have applied attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy to investigate the secondary structure of the pMMO. The results showed that ca. 60% of the amino acid residues were structured as alpha-helices. About 80% of the peptide residues were estimated to be protected from the amide (1)H/(2)H exchange during a 21 h exposure to (2)H(2)O. In addition, a significant portion of the protein was shown to be sequestered within the bilayer membrane, protected from trypsin proteolysis. The ATR-FTIR difference spectrum between the intact and the proteolyzed pMMO-enriched membranes revealed absorption peaks only in the spectral regions characteristic for unordered and beta-structures. These observations were corroborated by amino acid sequence analysis of the pMMO subunits using the program TransMembrane topology with a Hidden Markov Model: 15 putative transmembrane alpha-helices were predicted. Finally, an attempt was also made to model the three-dimensional folding of the protein subunits from the sequence using the Protein Fold Recognition Server based on the 3D Position Specific Scoring Matrix Method. The C-terminal solvent-exposed sequence (N255-M414) of the pMMO 45 kDa subunit was shown to match the beta-sheet structure of the multidomain cupredoxins. We conclude on the basis of this ATR-FTIR study that pMMO is an alpha-helical bundle with ca. 15 transmembrane alpha-helices embedded in the bilayer membrane, together with a water-exposed domain comprised mostly of beta-sheet structures similar to the cupredoxins.     

Engineering and rapid selection of a low-affinity glucose/galactose-binding protein for a glucose biosensor

Periplasmic expression screening is a selection technique used to enrich high-affinity proteins in Escherichia coli. We report using this screening method to rapidly select a mutated D-glucose/D-galactose-binding protein (GGBP) having low affinity to glucose. Wild-type GGBP has an equilibrium dissociation constant of 0.2 microM and mediates the transport of glucose within the periplasm of E. coli. The protein undergoes a large conformational change on binding glucose and, when labeled with an environmentally sensitive fluorophore, GGBP can relay glucose concentrations, making it of potential interest as a biosensor for diabetics. This use necessitates altering the glucose affinity of GGBP, bringing it into the physiologically relevant range for monitoring glucose in humans (1.7-33 mM). To accomplish this a focused library was constructed using structure-based site-saturation mutagenesis to randomize amino acids in the binding pocket of GGBP at or near direct H-bonding sites and screening the library within the bacterial periplasm. After selection, equilibrium dissociation constants were confirmed by glucose titration and fluorescence monitoring of purified mutants labeled site-specifically at E149C with the fluorophore IANBD (N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylene-diamine). The screening identified a single mutation A213R that lowers GGBP glucose affinity 5000-fold to 1 mM. Computational modeling suggested the large decrease in affinity was accomplished by the arginine side chain perturbing H-bonding and increasing the entropic barrier to the closed conformation. Overall, these experiments demonstrate the ability of structure-based site-saturation mutagenesis and periplasmic expression screening to discover low-affinity GGBP mutants having potential utility for measuring glucose in humans.     

Binding of the bioactive compound 5,7,4'-trihydroxy-6,3',5'-trimethoxyflavone to human serum albumin

5,7,4'-trihydroxy-6,3',5'-trimethoxyflavone is one of the bioactive components isolated from Artemisia plants possessing antitumor therapeutic activities. In this paper, its binding properties and binding sites located on human serum albumin (HSA) have been studied using UV absorption spectroscopy, fluorescence spectroscopy and Fourier transform infrared (FT-IR) spectra. The results of fluorescence titration revealed that 5,7,4'-trihydroxy-6,3',5'-trimethoxyflavone could strongly quench the intrinsic fluorescence of HSA by static quenching and there was only one class of binding sites on HSA for this drug. The binding constants at four different temperatures (289, 298, 310, and 318 K) were 1.93, 1.56, 1.22, and 0.93x10(5) L mol-1, respectively. The FT-IR spectra evidence showed that the protein secondary structure changed with reduction of alpha-helices about 27.6% at the drug to protein molar ratio of 3. The thermodynamic functions standard enthalpy change (DeltaH0) and standard entropy change (DeltaS0) for the reaction were calculated to be -18.70 kJ mol-1 and 36.62 J mol-1 K-1 according to the van't Hoff equation. These results and the molecular modeling study suggested that hydrophobic interaction was the predominant intermolecular force stabilizing the complex, and 5,7,4'-trihydroxy-6,3',5'-trimethoxyflavone could bind to the site I of HSA (the Warfarin Binding site).     

Membrane curvature and surface area per lipid affect the conformation and oligomeric state of HIV-1 fusion peptide: a combined FTIR and MD simulation study

Fourier-transformed infrared spectroscopy (FTIR) and molecular dynamics (MD) simulation results are presented to support our hypothesis that the conformation and the oligomeric state of the HIV-1 gp41 fusion domain or fusion peptide (gp41-FP) are determined by the membrane surface area per lipid (APL), which is affected by the membrane curvature. FTIR of the gp41-FP in the Aerosol-OT (AOT) reversed micellar system showed that as APL decreases from approximately 50 to 35 A2 by varying the AOT/water ratio, the FP changes from the monomeric alpha-helical to the oligomeric beta-sheet structure. MD simulations in POPE lipid bilayer systems showed that as the APL decreases by applying a negative surface tension, helical monomers start to unfold into turn-like structures. Furthermore, an increase in the applied lateral pressure during nonequilibrium MD simulations favored the formation of beta-sheet structure. These results provide better insight into the relationship between the structures of the gp41-FP and the membrane, which is essential in understanding the membrane fusion process. The implication of the results of this work on what is the fusogenic structure of the HIV-1 FP is discussed.