共查询到20条相似文献,搜索用时 15 毫秒
1.
《生物化学与生物物理学报:生物膜》2015,1848(2):721-730
Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) poses a serious public health hazard. The S2 subunit of the S glycoprotein of SARS-CoV carries out fusion between the virus and the host cells. However, the exact mechanism of the cell fusion process is not well understood. Current model suggests that a conformational transition, upon receptor recognition, of the two heptad core regions of S2 may expose the hydrophobic fusogenic peptide or fusion peptide for membrane insertion. Three regions of the S2 subunit have been proposed to be involved in cell–cell fusion. The N-terminal fusion peptide (FP, residues 770–788), an internal fusion peptide (IFP, residues 873–888) and the pre-transmembrane region (PTM, residues 1185–1202) demonstrated interactions with model lipid membranes and potentially involved in the fusion process. Here, we have determined atomic resolution structures of these three peptides in DPC detergent micelles by solution NMR. FP assumes α-helical conformation with significant distortion at the central Gly residues; enabling a close packing among sidechains of aromatic residues including W, Y and F. The 3-D structure of PMT is characterized by a helix–loop–helix with extensive aromatic interactions within the helices. IFP adopts a rather straight α-helical conformation defined by packing among sidechains of aromatic and aliphatic residues. Paramagnetic spin labeled NMR has demonstrated surface localization of PMT whereas FP and IFP inserted into the micelles. Collectively, data presented in this study will aid in understanding fusion mechanism of SARS-CoV. 相似文献
2.
Top D Read JA Dawe SJ Syvitski RT Duncan R 《The Journal of biological chemistry》2012,287(5):3403-3414
The p15 fusion-associated small transmembrane (FAST) protein is a nonstructural viral protein that induces cell-cell fusion and syncytium formation. The exceptionally small, myristoylated N-terminal ectodomain of p15 lacks any of the defining features of a typical viral fusion protein. NMR and CD spectroscopy indicate this small fusion module comprises a left-handed polyproline type II (PPII) helix flanked by small, unstructured N and C termini. Individual prolines in the 6-residue proline-rich motif are highly tolerant of alanine substitutions, but multiple substitutions that disrupt the PPII helix eliminate cell-cell fusion activity. A synthetic p15 ectodomain peptide induces lipid mixing between liposomes, but with unusual kinetics that involve a long lag phase before the onset of rapid lipid mixing, and the length of the lag phase correlates with the kinetics of peptide-induced liposome aggregation. Lipid mixing, liposome aggregation, and stable peptide-membrane interactions are all dependent on both the N-terminal myristate and the presence of the PPII helix. We present a model for the mechanism of action of this novel viral fusion peptide, whereby the N-terminal myristate mediates initial, reversible peptide-membrane binding that is stabilized by subsequent amino acid-membrane interactions. These interactions induce a biphasic membrane fusion reaction, with peptide-induced liposome aggregation representing a distinct, rate-limiting event that precedes membrane merger. Although the prolines in the proline-rich motif do not directly interact with membranes, the PPII helix may function to force solvent exposure of hydrophobic amino acid side chains in the regions flanking the helix to promote membrane binding, apposition, and fusion. 相似文献
3.
《生物化学与生物物理学报:生物膜》2018,1860(2):407-415
The lethal Coronaviruses (CoVs), Severe Acute Respiratory Syndrome-associated Coronavirus (SARS-CoV) and most recently Middle East Respiratory Syndrome Coronavirus, (MERS-CoV) are serious human health hazard. A successful viral infection requires fusion between virus and host cells carried out by the surface spike glycoprotein or S protein of CoV. Current models propose that the S2 subunit of S protein assembled into a hexameric helical bundle exposing hydrophobic fusogenic peptides or fusion peptides (FPs) for membrane insertion. The N-terminus of S2 subunit of SARS-CoV reported to be active in cell fusion whereby FPs have been identified. Atomic-resolution structure of FPs derived either in model membranes or in membrane mimic environment would glean insights toward viral cell fusion mechanism. Here, we have solved 3D structure, dynamics and micelle localization of a 64-residue long fusion peptide or LFP in DPC detergent micelles by NMR methods. Micelle bound structure of LFP is elucidated by the presence of discretely folded helical and intervening loops. The C-terminus region, residues F42-Y62, displays a long hydrophobic helix, whereas the N-terminus is defined by a short amphipathic helix, residues R4-Q12. The intervening residues of LFP assume stretches of loops and helical turns. The N-terminal helix is sustained by close aromatic and aliphatic sidechain packing interactions at the non-polar face. 15N{1H}NOE studies indicated dynamical motion, at ps-ns timescale, of the helices of LFP in DPC micelles. PRE NMR showed that insertion of several regions of LFP into DPC micelle core. Together, the current study provides insights toward fusion mechanism of SARS-CoV. 相似文献
4.
Carolina G Sarzedas Carla S Lima Maria A Juliano Luiz Juliano Ana Paula Valente Andrea T Da Poian Fabio C L Almeida 《Journal of peptide science》2008,14(4):429-435
Entry of enveloped animal viruses into their host cells always depends on a step of membrane fusion triggered by conformational changes in viral envelope glycoproteins. Vesicular stomatitis virus (VSV) infection is mediated by virus spike glycoprotein G, which induces membrane fusion at the acidic environment of the endosomal compartment. In a previous work, we identified a specific sequence in the VSV G protein, comprising the residues 145-164, directly involved in membrane interaction and fusion. In the present work we studied the interaction of pep[145-164] with membranes using NMR to solve the structure of the peptide in two membrane-mimetic systems: SDS micelles and liposomes composed of phosphatidylcholine and phosphatidylserine (PC:PS vesicles). The presence of medium-range NOEs showed that the peptide has a tendency to form N- and C-terminal helical segments in the presence of SDS micelles. Analysis of the chemical shift index indicated helix-coil equilibrium for the C-terminal helix under all conditions studied. At pH 7.0, the N-terminal helix also displayed a helix-coil equilibrium when pep[145-164] was free in solution or in the presence of PC:PS. Remarkably, at the fusogenic pH, the region of the N-terminal helix in the presence of SDS or PC:PS presented a third conformational species that was in equilibrium with the helix and random coil. The N-terminal helix content decreases pH and the minor beta-structured conformation becomes more prevalent at the fusogenic pH. These data point to a beta-conformation as the fusogenic active structure-which is in agreement with the X-ray structure, which shows a beta-hairpin for the region corresponding to pep[145-164]. 相似文献
5.
The six N-terminal transmembrane helices (N6) and the six C-terminal transmembrane helices (C6) in lactose permease, each containing a single Cys residue, were coexpressed, and cross-linking was studied. The proximity of paired Cys residues in helices V and VII, VIII, or X was studied by thiol-specific chemical cross-linking. The results demonstrate that Cys residues in the periplasmic half of helix V cross-link with Cys residues in the periplasmic half of helix VII. In contrast, no cross-linking is evident with paired Cys residues in the cytoplasmic halves of helices V and VII. Moreover, Cys residues on one entire face of helix V cross-link with Cys residues on one face of helix VIII. Finally, paired Cys residues at the cytoplasmic ends of helices V and X cross-link, but no cross-linking is observed when paired Cys residues are placed at the periplasmic ends of the two helices. Taken together, the results indicate that the periplasmic halves of helices V and VII are in close proximity and that the two helices tilt away from one another toward the cytoplasmic side of the membrane. Furthermore, helices V and VIII are in close proximity throughout their lengths and do not tilt appreciably with respect to one another, and helices V and X are in close proximity at the cytoplasmic but not at the periplasmic face of the membrane. 相似文献
6.
Sean T. Smrt Adrian W. Draney Justin L. Lorieau 《The Journal of biological chemistry》2015,290(1):228-238
The highly conserved N-terminal 23 residues of the hemagglutinin glycoprotein, known as the fusion peptide domain (HAfp23), is vital to the membrane fusion and infection mechanism of the influenza virus. HAfp23 has a helical hairpin structure consisting of two tightly packed amphiphilic helices that rest on the membrane surface. We demonstrate that HAfp23 is a new class of amphipathic helix that functions by leveraging the negative curvature induced by two tightly packed helices on membranes. The helical hairpin structure has an inverted wedge shape characteristic of negative curvature lipids, with a bulky hydrophobic region and a relatively small hydrophilic head region. The F3G mutation reduces this inverted wedge shape by reducing the volume of its hydrophobic base. We show that despite maintaining identical backbone structures and dynamics as the wild type HAfp23, the F3G mutant has an attenuated fusion activity that is correlated to its reduced ability to induce negative membrane curvature. The inverted wedge shape of HAfp23 is likely to play a crucial role in the initial stages of membrane fusion by stabilizing negative curvature in the fusion stalk. 相似文献
7.
T F Aripov S E Gasanov B A Salakhutdinov I A Rozenshtein F G Kamaev 《General physiology and biophysics》1989,8(5):459-473
The processes of membrane aggregation, permeability and fusion induced by cytotoxins from Central Asian cobra venom were investigated by studying optical density of liposome samples, permeability of liposome membranes for ferricyanide anions and exchange of lipid material between the membranes of adjacent liposomes. Cytotoxins Vc5 and Vc1 were found to induce aggregation of PC + CL and PC + PS liposomes. Cytotoxin Vc5 increased also the permeability of the liposomes for K3[Fe(CN)6] and enhanced their fusion. Cytotoxin Vc1 increased membrane permeability and enhanced fusion of PC + CL samples only. The changes in membrane permeability and fusion were found to occur within a single value of cytotoxin concentrations. The fusogenic properties of the cytotoxins studied are supposed to be due to the ability to dehydrate membrane surface and to destabilize the lipid bilayer structure. Fusion probability is largely defined by the phospholipid composition of the membranes. A model of interaction of cytotoxins with cardiolipin-containing membranes is offered. 相似文献
8.
Recombinant analogs of spider dragline silk proteins 1F9 and 2E12 are characterized by numerous repeats consisting of hydrophobic poly-Ala blocks and Gly-rich sequences with a substantial number of positively charged amino acid residues which suggest a pronounced ability to interact with negatively charged phospholipid membranes. Actually both proteins displayed substantial binding affinity towards lipid vesicles formed of acidic lipids as measured by fluorescence correlation spectroscopy (FCS) using rhodamine-labeled conjugates of the proteins. Both proteins did not induce liposome leakage, fusion or breakdown, but were able to bring about liposome aggregation. 1F9 was more active in the induction of liposome aggregation compared to 2E12. Interestingly, 2E12 markedly decreased the rate of calcium-induced liposome fusion. Circular dichroism data showed that binding of the proteins to negatively charged phosphatidylserine liposomes provoked transition from the left-handed helix of polyproline II (PPII) type to β-structures and α-helices. The data suggested predominantly surface location of membrane bound proteins without significant perturbation of their hydrophobic core. 相似文献
9.
Interactions of human lymphoblasts with targeted vesicles containing Sendai virus envelope proteins 总被引:2,自引:0,他引:2
We have studied the internalization of targeted fusogenic liposome content to leukemic T cells (CEM) in vitro. We describe a method for the covalent coupling of T101 antibody to the surface of liposomes and the incorporation of fusogenic viral protein into the liposome membrane. Hygromycin B, an impermeant inhibitor of protein synthesis, was encapsulated in the targeted fusogenic liposomes and delivered directly to the cytoplasm of leukemic T cells by fusion between the two membranes. The cytotoxic effect was measured by [3H]thymidine incorporation. We show that CEM are rapidly and specifically killed by the drug encapsulated in the targeted fusogenic liposomes. This effect is due to the binding of the liposome by means of the antibody and then to the fusion of the liposome with the targeted cell membrane, mediated by F protein. 相似文献
10.
A new class of fusion-associated small transmembrane (FAST) proteins encoded by the non-enveloped fusogenic reoviruses 总被引:9,自引:0,他引:9
The non-enveloped fusogenic avian and Nelson Bay reoviruses encode homologous 10 kDa non-structural transmembrane proteins. The p10 proteins localize to the cell surface of transfected cells in a type I orientation and induce efficient cell-cell fusion. Mutagenic studies revealed the importance of conserved sequence-predicted structural motifs in the membrane association and fusogenic properties of p10. These motifs included a centrally located transmembrane domain, a conserved cytoplasmic basic region, a small hydrophobic motif in the N-terminal domain and four conserved cysteine residues. Functional analysis indicated that the extreme C-terminus of p10 functions in a sequence-independent manner to effect p10 membrane localization, while the N-terminal domain displays a sequence-dependent effect on the fusogenic property of p10. The small size, unusual arrangement of structural motifs and lack of any homologues in previously described membrane fusion proteins suggest that the fusion-associated small transmembrane (FAST) proteins of reovirus represent a new class of membrane fusion proteins. 相似文献
11.
A molecular model for membrane fusion based on solution studies of an amphiphilic peptide from HIV gp41. 下载免费PDF全文
G. Fujii S. Horvath S. Woodward F. Eiserling D. Eisenberg 《Protein science : a publication of the Protein Society》1992,1(11):1454-1464
The mechanism of protein-mediated membrane fusion and lysis has been investigated by solution-state studies of the effects of peptides on liposomes. A peptide (SI) corresponding to a highly amphiphilic C-terminal segment from the envelope protein (gp41) of the human immunodeficiency virus (HIV) was synthesized and tested for its ability to cause lipid membranes to fuse together (fusion) or to break open (lysis). These effects were compared to those produced by the lytic and fusogenic peptide from bee venom, melittin. Other properties studied included the changes in visible absorbance and mean particle size, and the secondary structure of peptides as judged by CD spectroscopy. Taken together, the observations suggest that protein-mediated membrane fusion is dependent not only on hydrophobic and electrostatic forces but also on the spatial arrangement of the amino acid residues to form an amphiphilic structure that promotes the mixing of the lipids between membranes. A speculative molecular model is proposed for membrane fusion by alpha-helical peptides, and its relationship to the forces involved in protein-membrane interactions is discussed. 相似文献
12.
The six N-terminal transmembrane helices (N(6)) and the six C-terminal transmembrane helices (C(6)) in the lactose permease of Escherichia coli, each containing a single Cys residue, were coexpressed, and cross-linking was studied. The proximity of paired Cys residues in helices III (position 78, 81, 84, 86, 87, 88, 90, 93, or 96) and VII (position 227, 228, 231, 232, 235, 238, 239, 241, 243, 245, or 246) was examined by using iodine or two rigid homobifunctional thiol-specific cross-linking reagents with different lengths [N,N'-o-phenylenedimaleimide (o-PDM; 6 A) and N, N'-p-phenylenedimaleimide (p-PDM; 10 A)]. Cys residues in the periplasmic half of helix III (position 87, 93, or 96) cross-link to Cys residues in the periplasmic half of helix VII (position 235, 238, 239, 241, or 245). In contrast, no cross-linking is evident with paired Cys residues near the cytoplasmic ends of helices III (position 78 or 81) and VII (position 227, 228, 213, 232, or 235). Therefore, the periplasmic halves of helices III and VII are in close proximity, and the helices tilt away from each other toward the cytoplasmic face of the membrane. On the basis of the findings, a modified helix packing model for the permease is presented. 相似文献
13.
The transmembrane domains of membrane fusogenic proteins are known to contribute to lipid bilayer mixing as indicated by mutational studies and functional reconstitution of peptide mimics. Here, we demonstrate that mutations of a GxxxG motif or of Ile residues, that were previously shown to compromise the fusogenicity of the Vesicular Stomatitis virus G-protein transmembrane helix, reduce its backbone dynamics as determined by deuterium/hydrogen-exchange kinetics. Thus, the backbone dynamics of these helices may be linked to their fusogenicity which is consistent with the known over-representation of Gly and Ile in viral fusogen transmembrane helices. The transmembrane domains of membrane fusogenic proteins are known to contribute to lipid bilayer mixing. Our present results demonstrate that mutations of certain residues, that were previously shown to compromise the fusogenicity of the Vesicular Stomatitis virus G-protein transmembrane helix, reduce its backbone dynamics. Thus, the data suggest a relationship between sequence, backbone dynamics, and fusogenicity of transmembrane segments of viral fusogenic proteins. 相似文献
14.
Vonck J von Nidda TK Meier T Matthey U Mills DJ Kühlbrandt W Dimroth P 《Journal of molecular biology》2002,321(2):307-316
The sodium ion-translocating F(1)F(0) ATP synthase from the bacterium Ilyobacter tartaricus contains a remarkably stable rotor ring composed of 11 c subunits. The rotor ring was isolated, crystallised in two dimensions and analysed by electron cryo-microscopy. Here, we present an alpha-carbon model of the c-subunit ring. Each monomeric c subunit of 89 amino acid residues folds into a helical hairpin consisting of two membrane-spanning helices and a cytoplasmic loop. The 11 N-terminal helices are closely spaced within an inner ring surrounding a cavity of approximately 17A (1.7 nm). The tight helix packing leaves no space for side-chains and is accounted for by a highly conserved motif of four glycine residues in the inner, N-terminal helix. Each inner helix is connected by a clearly visible loop to an outer C-terminal helix. The outer helix has a kink near the position of the ion-binding site residue Glu65 in the centre of the membrane and another kink near the C terminus. Two helices from the outer ring and one from the inner ring form the ion-binding site in the middle of the membrane and a potential access channel from the binding site to the cytoplasmic surface. Three possible inter-subunit ion-bridges are likely to account for the remarkable temperature stability of I.tartaricus c-rings compared to those of other organisms. 相似文献
15.
Conformation of membrane‐bound proteins revealed by vacuum‐ultraviolet circular‐dichroism and linear‐dichroism spectroscopy 下载免费PDF全文
Koichi Matsuo Yasuyuki Maki Hirofumi Namatame Masaki Taniguchi Kunihiko Gekko 《Proteins》2016,84(3):349-359
Knowledge of the conformations of a water‐soluble protein bound to a membrane is important for understanding the membrane‐interaction mechanisms and the membrane‐mediated functions of the protein. In this study we applied vacuum‐ultraviolet circular‐dichroism (VUVCD) and linear‐dichroism (LD) spectroscopy to analyze the conformations of α‐lactalbumin (LA), thioredoxin (Trx), and β‐lactoglobulin (LG) bound to phosphatidylglycerol liposomes. The VUVCD analysis coupled with a neural‐network analysis showed that these three proteins have characteristic helix‐rich conformations involving several helical segments, of which two amphiphilic or hydrophobic segments take part in interactions with the liposome. The LD analysis predicted the average orientations of these helix segments on the liposome: two amphiphilic helices parallel to the liposome surface for LA, two hydrophobic helices perpendicular to the liposome surface for Trx, and a hydrophobic helix perpendicular to and an amphiphilic helix parallel to the liposome surface for LG. This sequence‐level information about the secondary structures and orientations was used to formulate interaction models of the three proteins at the membrane surface. This study demonstrates the validity of a combination of VUVCD and LD spectroscopy in conformational analyses of membrane‐binding proteins, which are difficult targets for X‐ray crystallography and nuclear magnetic resonance spectroscopy. Proteins 2016; 84:349–359. © 2016 Wiley Periodicals, Inc. 相似文献
16.
Ryan MA Akinbi HT Serrano AG Perez-Gil J Wu H McCormack FX Weaver TE 《Journal of immunology (Baltimore, Md. : 1950)》2006,176(1):416-425
Surfactant protein B (SP-B) is secreted into the airspaces with surfactant phospholipids where it reduces surface tension and prevents alveolar collapse at end expiration. SP-B is a member of the saposin-like family of proteins, several of which have antimicrobial properties. SP-B lyses negatively charged liposomes and was previously reported to inhibit the growth of Escherichia coli in vitro; however, a separate study indicated that elevated levels of SP-B in the airspaces of transgenic mice did not confer resistance to infection. The goal of this study was to assess the antimicrobial properties of native SP-B and synthetic peptides derived from the native peptide. Native SP-B aggregated and killed clinical isolates of Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, and group B streptococcus by increasing membrane permeability; however, SP-B also lysed RBC, indicating that the membranolytic activity was not selective for bacteria. Both the antimicrobial and hemolytic activities of native SP-B were inhibited by surfactant phospholipids, suggesting that endogenous SP-B may not play a significant role in alveolar host defense. Synthetic peptides derived from native SP-B were effective at killing both Gram-positive and Gram-negative bacteria at low peptide concentrations (0.15-5.0 microM). The SP-B derivatives selectively lysed bacterial membranes and were more resistant to inhibition by phospholipids; furthermore, helix 1 (residues 7-22) retained significant antimicrobial activity in the presence of native surfactant. These results suggest that the role of endogenous SP-B in host defense may be limited; however, synthetic peptides derived from SP-B may be useful in the treatment of bacterial pneumonias. 相似文献
17.
The N-terminal six transmenbrane helices (N6) and the C-terminal six transmembrane helices (C6) of the lactose permease, each containing a single-Cys residue, were coexpressed, and proximity was studied. Paired Cys residues in helices IV (positions 114, 116, 119, 122, 125, or 129) and VII (227, 231, 232, 234, 235, 238, 239, 242, 243, 245, or 246) or XI (350, 353, 354, 357, 361, or 364) were tested for cross-linking in the presence of two rigid homobifunctional thiol-specific cross-linkers, N,N'-o-phenylenedimaleimide (o-PDM; 6 A) and N,N'-p-phenylenedimaleimide (p-PDM; 10 A). Cys residues in the middle of helix IV (position 119 or 122) cross-link to Cys residues in the middle of helix VII (position 238, 239, 242, or 243). In contrast, no cross-linking is evident with paired Cys residues at either end of helix IV (position 114, 116, 125, or 129) or helix VII (position 227, 231, 232, 234, 235, 245, or 246). On the other hand, Cys residues in the cytoplasmic half of helix IV (position 125 or 129) cross-link with Cys residues in the cytoplasmic half of helix XI (position 350, 353, or 354), while paired Cys residues at the periplasmic ends of the two helices do not cross-link. The results indicate that helices IV and VII cross in a scissors-like manner with the cytoplasmic end of helix IV tilting toward helix XI. 相似文献
18.
Role of the simian virus 5 fusion protein N-terminal coiled-coil domain in folding and promotion of membrane fusion 下载免费PDF全文
Formation of a six-helix bundle comprised of three C-terminal heptad repeat regions in antiparallel orientation in the grooves of an N-terminal coiled-coil is critical for promotion of membrane fusion by paramyxovirus fusion (F) proteins. We have examined the effect of mutations in four residues of the N-terminal heptad repeat in the simian virus 5 (SV5) F protein on protein folding, transport, and fusogenic activity. The residues chosen have previously been shown from study of isolated peptides to have differing effects on stability of the N-terminal coiled-coil and six-helix bundle (R. E. Dutch, G. P. Leser, and R. A. Lamb, Virology 254:147-159, 1999). The mutant V154M showed reduced proteolytic cleavage and surface expression, indicating a defect in intracellular transport, though this mutation had no effect when studied in isolated peptides. The mutation I137M, previously shown to lower thermostability of the six-helix bundle, resulted in an F protein which was properly processed and transported to the cell surface but which had reduced fusogenic activity. Finally, mutations at L140M and L161M, previously shown to disrupt alpha-helix formation of isolated N-1 peptides but not to affect six-helix bundle formation, resulted in F proteins that were properly processed. Interestingly, the L161M mutant showed increased syncytium formation and promoted fusion at lower temperatures than the wild-type F protein. These results indicate that interactions separate from formation of an N-terminal coiled-coil or six-helix bundle are important in the initial folding and transport of the SV5 F protein and that mutations that destabilize the N-terminal coiled-coil can result in stimulation of membrane fusion. 相似文献
19.
The polar region consecutive to the HIV fusion peptide participates in membrane fusion 总被引:2,自引:0,他引:2
The fusion peptide of HIV-1 gp41 is formed by the 16 N-terminal residues of the protein. This 16-amino acid peptide, in common with several other viral fusion peptides, caused a reduction in the bilayer to hexagonal phase transition temperature of dipalmitoleoylphosphatidylethanolamine (T(H)), suggesting its ability to promote negative curvature in membranes. Surprisingly, an elongated peptide corresponding to the 33 N-terminal amino acids raised T(H), although it was more potent than the 16-amino acid fusion peptide in inducing lipid mixing with large unilamellar liposomes of 1:1:1 dioleoylphosphatidylethanolamine/dioleoylphosphatidylcholine/choleste rol. The 17-amino acid C-terminal fragment of the peptide can induce membrane fusion by itself, if it is anchored to a membrane by palmitoylation of the amino terminus, indicating that the additional 17 hydrophilic amino acids contribute to the fusogenic potency of the peptide. This is not solely a consequence of the palmitoylation, as a random peptide with the same amino acid composition with a palmitoyl anchor was less potent in promoting membrane fusion and palmitic acid itself had no fusogenic activity. The 16-amino acid N-terminal fusion peptide and the longer 33-amino acid peptide were labeled with NBD. Fluorescence binding studies indicate that both peptides bind to the membrane with similar affinities, indicating that the increased fusogenic activity of the longer peptide was not a consequence of a greater extent of membrane partitioning. We also determined the secondary structure of the peptides using FTIR spectroscopy. We find that the amino-terminal fusion peptide is inserted into the membrane as a beta-sheet and the 17 C-terminal amino acids lie on the surface of the membrane, adopting an alpha-helical conformation. It was further demonstrated with the use of rhodamine-labeled peptides that the 33-amino acid peptide self-associated in the membrane while the 16-amino acid N-terminal peptide did not. Thus, the 16-amino acid N-terminal fusion peptide of HIV inserts into the membrane and, like other viral fusion peptides, lowers T(H). In addition, the 17 consecutive amino acids enhance the fusogenic activity of the fusion peptide presumably by promoting its self-association. 相似文献
20.
The lactose permease of Escherichia coli was expressed in two fragments (split permease), each with a Cys residue, and cross-linking was studied. Split permease with a discontinuity in either loop II/III (N2C10permease) or loop VI/VII (N6C6permease) was used. Proximity of multiple pairs of Cys residues in helices I and XI or XII was examined by using three homobifunctional thiol-specific cross-linking reagents of different lengths and flexibilities (6 A, rigid; 10 A, rigid; 16 A, flexible) or iodine. Cys residues in the periplasmic half of helix I cross-link to Cys residues in the periplasmic half of helix XI. In contrast, no cross-linking is evident with paired Cys residues near the cytoplasmic ends of helices I and XI. Therefore, the periplasmic halves of helices I and XI are in close proximity, and the helices tilt away from each other towards the cytoplasmic face of the membrane. Cross-linking is also found with paired Cys residues near the middle of helices I and XII, but not with paired Cys residues near either end of the helices. Thus, helices I and XII are in close proximity only in the approximate middle of the membrane. Based on the findings, a modified helix packing model is proposed. 相似文献