Multiple states of beta-sheet peptide protegrin in lipid bilayers

Protegrin-1 (PG-1), a beta-sheet antimicrobial peptide, was studied in aligned lipid bilayers by oriented circular dichroism (OCD). All of its spectra measured in a variety of lipid compositions were linear superpositions of two primary basis spectra, indicating that PG-1 existed in two different states in membranes. We designated these as state S and state I. The state assumed by PG-1 was strongly influenced by lipid composition, peptide concentration, and hydration condition. We have previously reported that the helical peptides, alamethicin and magainin, also exhibit two distinct OCD basis spectra-one corresponding to surface adsorption with the helix parallel to the bilayer and the other with perpendicular transbilayer insertion. States S and I of PG-1 may correspond to the surface state and the insertion state of alamethicin, since they show a similar dependence on lipid composition, peptide concentration, and hydration condition. Nonoriented CD spectra obtained from vesicle, micelle, and solution preparations are not linear superpositions of the basis spectra of the states S and I. This indicates that a molecular orientation change alone is insufficient to describe the S left and right arrow I transition. Rather, a more complicated process is taking place, perhaps involving a change in the hydrogen bonding pattern of the backbone. Although the structural basis of the OCD spectra remains to be determined, the discovery of two distinct states can provide information about dynamic changes of PG-1 in membranelike environments, properties undoubtedly related to its antimicrobial and cytotoxic effects.     

Structure, orientation and affinity for interfaces and lipids of ideally amphipathic lytic LiKj(i=2j) peptides

The behavior of lytic ideally amphipathic peptides of generic composition LiKj(i=2j) and named LKn, n=i+j, is investigated in situ by the monolayer technique combined with the recently developed polarization modulation IR spectroscopy (PMIRRAS). A change in the secondary structure occurs versus peptide length. Peptides longer than 12 residues fold into alpha-helices at interfaces as expected from their design, while enough shorter peptides, from 9 down to 5 residues, form intermolecular antiparallel beta-sheets. Analysis of experimental and calculated PMIRRAS spectra in the amide I and II regions show that peptides are flat oriented at the interfaces. Structures and orientation are preserved whatever the nature of the interface, air/water or DMPC monolayer, and the lateral pressure. Peptide partition constants, KaffPi, are estimated from isobar surface increases of DMPC monolayers. They strongly increase when Pi decreases from 30 mN/m to 8 mN/m and they vary with peptide length with an optimum for 12 residues. This non-monotonous dependence fits with data obtained in bilayers and follows the hemolytic activity of the peptides. Lipid perturbations due to peptide insertion essentially detected on the PO4- and CO bands indicate disorder of the lipid head groups. Lysis induced on membranes by such peptides is proposed to first result from their flat asymmetric insertion.     

Characterization of the conformations of antigenic peptides of protein lactate dehydrogenase (LDH-C4) by electrospray ionization mass spectrometry

The conformations of several rationally designed antigenic peptides that mimic, to varying degrees, an antibody-binding region of protein lactate dehydrogenase isozyme (LDH-C4) are investigated by deuterium/hydrogen exchange and electrospray ionization mass spectrometry (ESI-MS). The approach involves monitoring the reverse-exchange of deuterium, incorporated at the labile sites in the peptides, with hydrogen as a function of time by ESI-MS. Idealized forms of a segment of the native antigen are shown to be more conformationally restricted than the native peptide based on level of deuterium that remains incorporated at the labile sites over time. From the number of amide groups of the peptide backbone that retain deuterium, estimates of the helical content of each peptide have been measured that are in close agreement with those determined by Fourier transform infrared (FTIR) spectroscopy in separate experiments. A single amino acid substitution in the idealized helical construct results in a conformational change easily detected by the deuterium exchange ESI-MS method. The approach is shown to be a viable method for characterizing the conformations of protein antigens at the local level and for screening the conformations of antigenic peptides designed to elicit optimal immune responses.     

Structural properties of the putative fusion peptide of fertilin, a protein active in sperm-egg fusion, upon interaction with the lipid bilayer

We recently demonstrated that a peptide representing the putative fusion domain of fertilin, a surface membrane protein of sperm involved in sperm-egg fusion, induces fusion of large unilamellar vesicles containing negatively charged lipids [Martin, I., and Ruysschaert, J. M. (1997) FEBS Lett. 405, 351-355]. In the present work, we demonstrate that increasing the concentration in negatively charged lipids strongly enhances the binding of the fertilin fusion peptide to the membrane, suggesting that electrostatic attractions play a crucial role in the binding process. While no significant change of the secondary structure content is observed by increasing the amounts of negatively charged lipids in the bilayer, the orientation of the alpha-helix changes from a parallel to an oblique orientation in the membrane. This topological change is confirmed by amide II hydrogen/deuterium exchange measurements that monitor the accessibility of the peptide to the water medium. Differential scanning calorimetry data also suggest that the fertilin fusion peptide lowers the bilayer to hexagonal phase transition temperature of model membranes composed of mixtures of dipalmitoleoylphosphatidylethanolamine and 1-palmitoyl-2-oleoylphosphatidylserine and therefore promotes negative curvature in lipid vesicles. A comparison of the biophysical properties and the membrane-perturbing activities of fertilin and of viral fusion peptides is discussed in terms of sperm-egg fusion and virus cell fusion.     

Characterisation of the processing by human neutral endopeptidase 24.11 of GLP-1(7-36) amide and comparison of the substrate specificity of the enzyme for other glucagon-like peptides

The post-secretory processing of the potent insulinotropic peptide hormone, GLP-1(7-36)amide, probably involves one or more of a small group of membrane-bound ectopeptidases. Reported here, is the characterisation of the endoproteolysis of human GLP-1(7-36)amide by the recombinant human form of neutral endopeptidase (NEP) 24.11, which is one of the best characterised and widely-distributed of ectopeptidases and is involved in the processing of other peptide hormones. The products of the limited endoproteolysis were characterised by mass and primary structure following fractionation using high performance liquid chromatography. The rate of this endoproteolysis by NEP 24.11 was estimated and compared to that of GLP-1(7-36)amide-related peptides. GLP-1(7-36)amide appears to be good substrate for NEP 24.11 with most, but not all potential target bonds being cleaved. Also, the structurally-related peptides, secretin and glucagon appear to be good substrates whereas GIP and exendin-4 are very poor substrates. That the GLP-1(7-36)amide super-agonist, exendin-4 is a poor substrate for NEP 24.11 is significant for the possible use of this peptide as a prototype for the development of clinically-useful peptide agonists. Further studies should reveal whether NEP 24.11 is important for the metabolic clearance of GLP-1(7-36)amide and will be highly relevant for the attempts to realise the suggested therapeutic value of GLP-1(7-36)amide.     

Effect of the N-terminal glycine on the secondary structure, orientation, and interaction of the influenza hemagglutinin fusion peptide with lipid bilayers

The amino-terminal segment of the membrane-anchored subunit of influenza hemagglutinin (HA) plays a crucial role in membrane fusion and, hence, has been termed the fusion peptide. We have studied the secondary structure, orientation, and effects on the bilayer structure of synthetic peptides corresponding to the wild-type and several fusogenic and nonfusogenic mutants with altered N-termini of the influenza HA fusion peptide by fluorescence, circular dichroism, and Fourier transform infrared spectroscopy. All peptides contained segments of alpha-helical and beta-strand conformation. In the wild-type fusion peptide, 40% of all residues were in alpha-secondary and 30% in beta-secondary structures. By comparison, the nonfusogenic peptides exhibited larger beta/alpha secondary structure ratios. The order parameters of the helices and the amide carbonyl groups of the beta-strands of the wild-type fusion peptide were measured separately, based on the infrared dichroism of the respective absorption bands. Order parameters in the range 0.1-0.7 were found for both segments of the wild-type peptide, which indicates that they are most likely aligned at oblique angles to the membrane normal. The nonfusogenic but not the fusogenic peptides induced splitting of the infrared absorption band at 1735 cm(-1), which is assigned to stretching vibrations of the lipid ester carbonyl bond. This splitting, which reports on an alteration of the hydrogen bonds formed between the lipid ester carbonyls and water and/or hydrogen-donating groups of the fusion peptides, correlated with the beta/alpha ratio of the peptides, suggesting that unpaired beta-strands may replace water molecules and hydrogen-bond to the lipid ester carbonyl groups. The profound structural changes induced by single amino acid replacements at the extreme N-terminus of the fusion peptide further suggest that tertiary or quaternary structural interactions may be important when fusion peptides bind to lipid bilayers.     

Membrane fusion induced by 11-mer anionic and cationic peptides: a structure-function study

We recently demonstrated that an amphipathic net-negatively charged peptide consisting of 11 amino acids (WAE 11) strongly promotes fusion of large unilamellar liposomes (LUV) when anchored to a liposomal membrane [Pecheur, E. I., Hoekstra, D., Sainte-Marie, J., Maurin, L., Bienvenue, A., and Philippot, J. R. (1997) Biochemistry 36, 3773-3781]. To elucidate a potential relationship between peptide structure and its fusogenic properties and to test the hypothesis that specific structural motifs are a prerequisite for WAE-induced fusion, three 11-mer WAE-peptide analogues (WAK, WAEPro, and WAS) were synthesized and investigated for their structure and fusion activity. Structural analysis of the synthetic peptides by infrared attenuated total reflection spectroscopy reveals a distinct propensity of each peptide toward a helical structure after their anchorage to a liposomal surface, emphasizing the importance of anchorage on conveying a secondary structure, thereby conferring fusogenicity to these peptides. However, whereas WAE and WAK peptides displayed an essentially nonleaky fusion process, WAS- and WAEPro-induced fusion was accompanied by substantial leakage. It appears that peptide helicity as such is not a sufficient condition to convey optimal fusion properties to these 11-mer peptides. Studies of changes in the intrinsic Trp fluorescence and iodide quenching experiments were carried out and revealed the absence of migration of the Trp residue of WAS and WAEPro to a hydrophobic environment, upon their interaction with the target membranes. These results do not support the penetration of both peptides as their mode of membrane interaction and destabilization but rather suggest their folding along the vesicle surface, posing them as surface-seeking helixes. This is in striking contrast to the behavior observed for WAE and WAK, for which at least partial penetration of the Trp residue was demonstrated. These results indicate that subtle differences in the primary sequence of a fusogenic peptide could induce dramatic changes in the way the peptide interacts with a bilayer, culminating in equally drastic changes in their functional properties. The data also reveal a certain degree of sequence specificity in WAE-induced fusion.     

Structure of the membrane-bound form of the pore-forming domain of colicin A: a partial proteolysis and mass spectrometry study

The ion-channel-forming thermolytic fragment (thA) of colicin A binds to negatively charged vesicles and provides an example of the insertion of a soluble protein into a lipid bilayer. The soluble structure is known and consists of a 10-helix bundle containing a hydrophobic helical hairpin. In this study, partial proteolysis and mass spectrometry were used to determine the accessible sites to proteolytic attack by trypsin and alpha-chymotrypsin in the thA fragment in its membrane-bound state. Electrospray mass spectrometry was quite an efficient method for the identification of the cleavage products, even with partially purified peptide mixtures and with only few controls by N-terminal sequencing. This work confirms that a major part of the peptide chain lies at the membrane surface and that even the hydrophobic hairpin is not protected by the lipid bilayer from proteolytic degradation. In the absence of a membrane potential, the hydrophobic hairpin in the colicin A membrane-bound form seems not fixed in a transmembrane orientation.     

Paramyxovirus fusion protein: characterization of the core trimer, a rod-shaped complex with helices in anti-parallel orientation

The fusion (F) protein of the paramyxovirus SV5 contains two heptad repeat regions, HRA adjacent to the fusion peptide and HRB proximal to the transmembrane domain. Peptides, N-1 and C-1, respectively, corresponding to these heptad repeat regions form a thermostable, alpha-helical trimer of heterodimers (S. B. Joshi, R. E. Dutch, and R. A. Lamb (1998). Virology 248, 20-34). Further characterization of the N-1/C-1 complex indicated that the C-1 peptides, which are predicted to residue on the outside of the complex, are resistant to digestion by several proteases when present in the complex. Only proteinase K digested most of the C-1 peptide, though the small remaining protease protected fragment of C-1 confers extreme thermostability on the proteinase-K-resistant N-1 trimeric coiled-coil. Carboxypeptidase Y digestion of the N-1/C-1 complex indicates that the C-1 peptides associate in an antiparallel orientation relative to the N-1 peptides. Electron microscopy of the N-1/C-1 complex showed a rod-shaped complex with an average length of 9.7 nm, consistent with all of N-1 existing as an alpha helix. Mutations at heptad repeat a and d residues of N-1, positions that are predicted to point inward to the center of the N-1 trimeric coiled-coil, were found to have varying effects as analyzed by circular dichroism measurements. The mutation I137M did not affect the helical structure of the isolated N-1 peptide but did affect the thermostability of the N-1/C-1 complex. Mutations L140M and L161M perturbed the helical structure formed by N-1 in isolation but did not affect formation of a thermostable N-1/C-1 complex. Finally, a peptide, SV5 F 255-293, corresponding to a proposed leucine zipper region, was analyzed for effects on N-1, C-1, or the N-1/C-1 complex. Circular dichroism analysis demonstrated that while the presence of peptide 255-293 increased the helical signal from either N-1 or the N-1/C-1 complex, no change in thermostability was observed, indicating that this region is not a component of the final, most stable core of the F protein.     

The effects of NGF and sensory nerve stimulation on collateral sprouting and gene expression in adult sensory neurons

Cerebroside sulfate activator protein (CSAct or saposin B) is one of a group of heat stable, low-molecular-weight proteins that appear to share a common structural motif. These have been referred to as saposin-like proteins and are thought to share a multiple amphipathic helical barrel structure with a conserved pattern of disulfide linkages. Porcine kidney CSAct was prepared in high purity and consisted of three major glycosylated subforms. The protein was studied by physical-chemical methods and evaluated by various methods for structural prediction. All suggest that CSAct has high amounts of alpha-helical conformation and little if any beta-sheet. Circular dichroism (CD) studies indicate 45-50% helical conformation depending on buffer and temperature. There was only a moderate loss in helical content with increasing temperature and no indication of thermal denaturation. Fourier transform infrared spectroscopy (FTIR) measurements on deuterium hydrated self-films also indicated a predominantly helical structure. Helical axis orientation was investigated by both oriented CD and FTIR dichroism, which suggested that the helical axes were roughly parallel and oriented along the axis of the surface on which the self-films had been deposited. One-dimensional nuclear magnetic resonance spectra showed large chemical shift dispersion, indicating a defined tertiary structure with little variation between 6 and 85 degrees C. NOESY spectra failed to show the strong NOE cross peaks expected for a highly helical conformation. This may indicate short-term conformational flexibility within the helices or molecular aggregation at the high protein concentrations employed. These observations are consistent with the 3-4-helix bundle motif suggested for saposin-like proteins by various predictive algorithms.     

Molecular and cellular actions of chronic electroconvulsive seizures

Reducing a CO to a CH2 moiety in a peptide bond destroys the ability of the peptide link to act as a proton acceptor in a hydrogen-bonded structure. Here, this modification is introduced into different positions of the helical peptide, acetyl-WGG(RAAAA)4R-amide, and the melting of these peptides is followed using CD. Effects of this modification on helical peptides are compared to our previous N-methylation studies [C. F. Chang and M. H. Zehfus (1996) Biopolymers, Vol. 40, pp. 609-616]. While the experiments were designed to remove the same hydrogen bond from the peptide, no consistent results are obtained between these two modifications. This result suggests that these modifications not only break the backbone hydrogen bonds, but also involve other destabilizing effects. When our data is analyzed using different helix-coil transition models, the results show that as the models increase in complexity the energy associated with a single residue modification increases. Unfortunately, the most detailed dichroic model, which should best describe this system, works for only one peptide. Apparently, the models need to be further improved to better mimic our system.     

Peptide hydrophobicity controls the activity and selectivity of magainin 2 amide in interaction with membranes

The magainins are antibacterial peptides from the skin of Xenopus laevis. They show a broad range of activity against prokaryotic cells but lyse eukaryotic cells poorly. To elucidate the influence of peptide hydrophobicity on membrane activity and selectivity, we designed and synthesized analogs of magainin 2 amide with slightly varying hydrophobicities but retained hydrophobic moment, peptide charge, and angle subtended by the hydrophilic helix region. Circular dichroism investigations of the peptides revealed that all peptides investigated adopt an alpha-helical conformation when bound to phospholipid vesicles. Dye-releasing experiments from vesicles of phosphatidylglycerol (PG) showed that the membrane-permeabilizing activity of the analogs is not influenced by peptide hydrophobicity. In contrast, the permeability-enhancing activity on vesicles bearing high amounts of phosphatidylcholine (PC) increases drastically with enhanced peptide hydrophobicity, resulting in a reduced selectivity of more hydrophobic analogs for negatively charged membranes. Likewise, the peptide affinity to PC-rich membranes increases in the order of hydrophobicity. Correlation of peptide binding and membrane permeabilization of PC/PG (3:1) vesicles revealed that the observed differences in peptide activity on membranes of low negative surface charge are mainly caused by the different binding affinities. The antibacterial and hemolytic activity of the peptides increases with enhanced hydrophobicity. A strong correlation was found between the hemolytic effect and the bilayer-permeabilizing activity against PC-rich vesicles. Whereas the antibacterial specificity of the more hydrophobic analogs is retained for Escherichia coli, the specificity for Pseudomonas aeruginosa decreases with increasing hydrophobicity.     

Role of autologous bone marrow transplantation as consolidation therapy in acute promyelocytic leukemia patients in complete remission

Recent studies in the field of de novo protein design have focused on the construction of native-like structures. Here we describe the design and characterization of an isoleucine zipper peptide intended to form a parallel triple-stranded coiled coil. To obtain the native-like structural uniqueness, the hydrophobic interface of the peptide consists of beta-branched Ile residues for complementary side chain packing. The peptide forms a stable triple-stranded coiled coil, as determined by circular dichroism and sedimentation equilibrium analyses. A fluorescence quenching assay after the incorporation of acridine revealed a parallel orientation of the peptides. The structural uniqueness of the coiled coil was confirmed by proton-deuterium amide hydrogen exchange and hydrophobic dye binding. The peptide contains amide protons with hydrogen exchange rates that are approximately an order of magnitude slower than those expected if the exchange occurred via global unfolding. A hydrophobic dye does not bind to the peptide. These results strongly suggest that the peptide folds into a well-packed structure that is very similar to the native state of a natural protein. Thus, Ile residues in the hydrophobic interface can improve the side chain packing, which can impart native-like structural uniqueness to the designed coiled coil.     

Recall of old and recent information

The potential therapeutic use of peptides to activate or anergize specific T cells is seriously limited by their susceptibility to proteolytic degradation. Classically, peptides are stabilized by incorporation of non-natural modifications including main chain modifications. In the case of MHC II-restricted peptides, the peptide backbone actively participates to the interaction with the MHC molecule and hence may preclude the peptidomimetic approach. We thus investigated whether a single amide bond modification influenced the peptide capacity to bind to a MHC II molecule and to stimulate specific T cells. Twenty pseudopeptide analogs of the I-Ed binder 24-36 peptide, whose sequence was derived from a snake neurotoxin, were obtained by replacing each amide bond of the peptide central part, by either a reduced psi[CH2-NH] or N-methylated psi[CO-NMe] peptide bond. In agreement with the major interacting role played by the peptide backbone, several peptides displayed a low, if any, capacity to bind to the MHC II molecule and did not lead to T cell stimulation. However, one-third of the peptides were almost as active as the 24-36 peptide in I-Ed binding assays and one-fifth in T cell stimulation assays. Among them, two pseudopeptides displayed native-like activity. Good binders were not necessarily good at stimulating T cells, demonstrating that main chain modification also affected T cell recognition. We thus showed that a peptidomimetic approach could create a new type of MHC II ligand to control T cell responses.     

Contribution of the hydrophobicity gradient of an amphipathic peptide to its mode of association with lipids

A class of peptides that associate with lipids, known as oblique-orientated peptides, was recently described [Brasseur R., Pillot, T., Lins, L., Vandekerckhove, J. & Rosseneu, M. (1997) Trends Biochem. Sci. 22, 167-171]. Due to an asymmetric distribution of hydrophobic residues along the axis of the alpha-helix, such peptides adopt an oblique orientation which can destabilise membranes or lipid cores. Variants of these oblique peptides, designed to have an homogeneous distribution of hydrophobic and hydrophilic residues along the helical axis, are classified as regular amphipathic peptides. These peptides are expected to lie parallel to the polar/apolar interface with their hydrophobic residues directed towards the apolar and their hydrophilic residues towards the polar phase. An hydrophobic, oblique-orientated peptide was identified at residues 56-68 in the sequence of the lecithin-cholesterol acyltransferase (LCAT), enzyme. This peptide is predicted to penetrate a lipid bilayer at an angle of 40 degrees through its more hydrophobic C-terminal end and thereby induce the destabilisation of a membrane or a lipid core. The LCAT-(56-68) wild-type peptide was synthesised together with the LCAT-(56-68, 0 degrees) variant, in which the hydrophobicity gradient was abolished through residue permutations. In two other variants, designed to keep their oblique orientation, the W61 residue was shifted either towards the more hydrophilic N-terminal at residue 57, or to position 68 at the hydrophobic C-terminal end of the peptide. Peptide-induced vesicle fusion was demonstrated by fluorescence measurements using pyrene-labeled vesicles and by monitoring of vesicle size by gel filtration. The interaction between peptides and lipids was monitored by measurement of the intrinsic tryptophan fluorescence emission of the peptides. Fluorescence polarisation measurements, using diphenyl hexatriene, were carried out to follow changes in the lipid fluidity. The LCAT-(56-68) wild-type peptide and the two oblique variants, induced fusion of unilamellar dimyristoylglycerophosphocholine vesicles. Tryptophan fluorescence emission measurements showed a 12-14 nm blue shift upon addition of the wild-type peptide and of the W61-->68 variant to lipids, whereas the fluorescence of the W61-->57 variant did not change significantly. This observation supports the insertion of the more hydrophobic C-terminal residues into the lipid phase, as predicted by the theoretical calculations. In contrast, the 0 degrees variant peptide had no fusogenic activity, and it associated with lipids to form small discoidal lipid/peptide complexes. The phospholipid transition temperature was decreased after addition of the wild-type, the W61-->68 and W61-->57 fusogenic peptides, whereas the opposite effect was observed with the 0 degrees variant. The behaviour of the wild-type and variant LCAT-(56-68) peptides stresses the contribution of the hydrophobicity gradient along the axis of an amphipathic peptide to the mode of association of this peptide with lipids. This parameter consequently influences the structural modifications occurring to lipids upon association with amphipathic peptides.     

Orientation of fusion-active synthetic peptides in phospholipid bilayers: determination by Fourier transform infrared spectroscopy

A group of synthetic peptides having an amino acid sequence related to the N-terminal region of the influenza virus hemagglutinin HA-2 chain can induce phospholipid membrane fusion in a pH-dependent manner. These peptides bind to membranes to form alpha-helices even at pH's where no fusion activity is seen. We determined the orientation of these alpha-helical peptides in lipid multibilayers using attenuated total reflection infrared spectroscopy and found that the peptide alpha-helices took a preferential orientation, the helix axis being about 70 degrees from the normal of the membrane plane, or in other words rather parallel to the membrane plane. The orientation was almost independent of pH and a modification of the N-terminal amino group which reduced the fusion activity of the peptides. The determination was carried out for peptides in lipid multibilayers in dry or hydrated (membranes equilibrated with D2O vapor) conditions. Although a slight decrease in the helix orientation angle from the membrane normal was noticed for a hydrated system, the difference between the results for dry and hydrated conditions was small.     

Synthetic peptides derived from the fourth domain of CD4 antagonize off function and inhibit T cell activation

We have developed synthetic peptide analogs to analyze novel surface structures of the human CD4 protein potentially involved in T cell activation. Linear and cyclic peptides derived from the FG and CC' loops of the membrane proximal fourth domain of CD4 displayed inhibitory activities in a CD4-dependent immunological assay. These results suggest that the fourth domain of CD4 plays an important role in T cell activation. In addition, we report the synthesis of a highly stable CD4 peptide analog cyclized by the formation of an amide bond between amino and carboxyl termini. Serum stability studies showed that this main-chain cyclic CD4 peptide was highly resistant to proteolytic degradation while the linear and disulfide cyclic peptides were much less stable. The strategy of main chain cyclization of CD4 peptides may represent a promising approach to generate proteolytically stable, orally active immunoregulatory agents.     

Crystal structure of the simian immunodeficiency virus (SIV) gp41 core: conserved helical interactions underlie the broad inhibitory activity of gp41 peptides

The gp41 subunit of the envelope protein complex from human and simian immunodeficiency viruses (HIV and SIV) mediates membrane fusion during viral entry. The crystal structure of the HIV-1 gp41 ectodomain core in its proposed fusion-active state is a six-helix bundle. Here we have reconstituted the core of the SIV gp41 ectodomain with two synthetic peptides called SIV N36 and SIV C34, which form a highly helical trimer of heterodimers. The 2.2 A resolution crystal structure of this SIV N36/C34 complex is very similar to the analogous structure in HIV-1 gp41. In both structures, three N36 helices form a central trimeric coiled coil. Three C34 helices pack in an antiparallel orientation into highly conserved, hydrophobic grooves along the surface of this coiled coil. The conserved nature of the N36-C34 interface suggests that the HIV-1 and SIV peptides are functionally interchangeable. Indeed, a heterotypic complex between HIV-1 N36 and SIV C34 peptides is highly helical and stable. Moreover, as with HIV-1 C34, the SIV C34 peptide is a potent inhibitor of HIV-1 infection. These results identify conserved packing interactions between the N and C helices of gp41 and have implications for the development of C peptide analogs with broad inhibitory activity.     

Investigation of secondary and tertiary structural changes of cytochrome c in complexes with anionic lipids using amide hydrogen exchange measurements: an FTIR study

The structure of cytochrome c bound to anionic lipid membranes composed of dimyristoyl, dipalmitoyl, or dioleoyl phosphatidylglycerols, or of bovine heart cardiolipin, has been investigated by Fourier transform infrared spectroscopy. Only small changes in secondary structure, as registered by the amide I band of cytochrome c, were observed upon binding at temperatures below that of denaturation of the protein, and these were not coupled to the thermotropic phase transitions of the lipid. The denaturation temperature of the protein decreased by approximately 25-30 degrees upon binding, in a progression which correlated with that of the lipid phase transition temperatures, being approximately 7 degrees lower for complexes with dioleoyl than with dipalmitoyl phosphatidylglycerol. Large changes in the amide proton exchange characteristics, as monitored by the spectral shifts in the amide I band of the protein in D2O, were observed on binding cytochrome c to the lipid membranes. For the slowly exchanging population, the amide deuteration rates of the free protein were nearly independent of temperature, whereas those of the bound protein increased by up to two orders of magnitude over the temperature range from 10 to 40 degrees C. In addition, the extent of exchange differed between the bound and unbound protein. A structural transition in the bound protein was detected as a discontinuous step in Arrhenius plots of the deuterium exchange rates which occurred at a temperature in the region of 22 to 29 degrees C, depending on the lipid, far below that of denaturation. The temperature of this transition was determined by the physical state of the lipid, being 7 degrees lower for the lipids in the fluid state than for those in the gel state, and, for complexes with dimyristoyl phosphatidylglycerol, occurred at an intermediate temperature, being controlled by the lipid chain-melting transition at 27-28 degrees C. These results provide evidence for a coupling of the tertiary structure of the membrane-bound protein with the physical state of the membrane lipids.     

Secondary structure analysis of individual transmembrane segments of the nicotinic acetylcholine receptor by circular dichroism and Fourier transform infrared spectroscopy

Circular dichroism (CD) and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy are used to establish the secondary structure of peptides containing one or more transmembrane segments (M1-M4) of the Torpedo californica nicotinic acetylcholine receptor (AChR). Peptides containing the M2-M3 and M1-M2-M3 transmembrane segments of the AChR beta-subunit and the M4 segment of the alpha- and gamma-subunits were isolated from proteolytic digests of receptor subunits, purified, and reconstituted into lipid vesicles. For each peptide, an amide I vibrational frequency centered between 1650 and 1656 cm-1 and negative CD absorption bands at 208 and 222 nm indicate that the peptide is largely alpha-helical. In addition, the CD spectrum of a tryptic peptide of the alpha-subunit containing the M1 segment is also consistent with a largely alpha-helical structure. However, secondary structure analysis of the alpha-M1 CD spectrum indicates the presence of other structures, suggesting that the M1 segment may represent either a distorted alpha-helix, likely the consequence of several proline residues, or may not be entirely alpha-helical. Overall, these findings are consistent with studies that indicate that the transmembrane region of the AChR comprises predominantly, if not exclusively, membrane-spanning alpha-helices.