Structure and membrane interaction of the internal fusion peptide of avian sarcoma leukosis virus Shu-Fang Cheng, Cheng-Wei Wu, Eric Assen B Kantchev and Ding-Kwo Chang Institute of Chemistry, Academia Sinica, Taipei, Taiwan, Republic of China The structure and membrane interaction of the internal fusion peptide (IFP) fragment of the avian sarcoma and leucosis virus (ASLV) envelope glycoprotein was studied by an array of biophysical methods. The peptide w as found to induce lipid mixing of ves icles more stron gly than the f usion peptide derived from the N-terminal fusion peptide of influenza virus (HA2-FP). It was observed that the helical structure was enhanced in association with the model membranes, p articularly in the N -terminal portion of the peptide. According to the infrared study, the peptide inserted into the membrane in a n oblique orientation, but less deeply than the influenza HA2-FP. Analysis of NMR d ata in sodium dodecyl sulfate micelle suspension revealed that Pro13 of the peptide was located near the micelle–water interface. A type II b-turn was deduced from NMR data for the peptide in aqueous medium, demonstrating a c onforma- tional flexibility of t he IFP in a nalogy to the N -terminal FP such as that of gp41. A loose and multim odal self-assembly was deduced from the rhodamine fluorescence self-quench- ing experiments for the peptide bound to the membrane bilayer. Oligomerization o f the peptide and its variants can also be observed in the electrophoretic experiments, sug- gesting a property in common with other N-terminal FP of class I fusion pr oteins. Keywords: membrane fusion; conformational change; insertion d epth; self-assembly; fluorescence self-quenching. Entry of e nveloped viruses into the host cells is mediated by the viral envelope glycoproteins [1], which in most cases are cleave d by proteolysis to yield the transmem- brane (TM) [ 2,3] subunit responsible for membrane fusion and the surface (SU) subunit f or receptor binding. For a majority of the class I fusion proteins, a region in t he TM protein crucial for binding to and destabilizing target membranes, termed fusion peptide (FP), is located a t the N-terminal region, while others have the internal fusion peptide (IFP) domain [ 4]. Avain sarcoma/leucosis virus (ASLV) is a prototype retrovirus [5], the envelope glycoprotein of which uses IFP for fusion to target cells [6,7]. A proline is often found near the centre of many of the viral IFP sequences [1]. Delos et al. [8] have shown that the central proline of the FP of ASLV subtype A plays important roles in forming a native e nvelope protein (EnvA) structure a nd in membrane fusion. It is thought that the envelope protein undergo es conformational change triggered by i ts binding to the receptor on the target cell surface (e.g. Tva f or ASLV-A), exposing the hydrophobic FP domain to destabilize t he cell membrane preceding the membrane fusion [9] similar to influenza haemagglutinin and HIV-1 gp41. As the majority of studies were performed on the N-terminal FP, it would be of interest to compare the structure of the internal FP and i ts interaction w ith membrane bilayer, including in particular the structural influence of proline. Consistent with other c lass I viral fus ion proteins, the IFP o f ASLV inserts into the membrane primarily as a h elix in contrast to the I FP of class II fusion protein which uses a Ôcd loopÕ to insert into the target membrane in the fusion process [10,11]. In the following, a variety of physical properties of the putative IFP of ASLV are reported and differences between N-terminal and internal FP a re compared. The pH dependence of s ome o f the properties is discussed in regard to the experimental observation that ASLV induced hemifusion, but not complete fusion, at neutral pH [12]. Experimental procedures All chemicals and solvents were used without further purification. N-a-(9-Fluorenylmethoxycarbonyl) ( Fmoc)- protected amino acids were products of Anaspec (San Jose, CA, USA) or Bachem (Bubendorf, Switzerland). 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and Correspondence to D K. Chang, Institute of Chemistry, Academia Sinica, Taipei, Taiwan 115, Republic of China. Fax: + 886 2 27831237, Tel.: + 886 2 2 7 898594, E-mail: dkc@chem.sinica.edu.tw Abbreviations: ASLV, avain/sarcoma leucosis virus; ASLV-A, ASLV subtype A; ATR-FTIR, attenuated total reflectance-FTIR; DG, dis- tance geometry; DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocho- line; DMPG, 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol; EnvA, native envelope protein; FP, fusion peptide; HA2-FP, N-terminal fusion peptide of influenza virus; IFP, internal fusion peptide; NBD, 7-nitrobenz-2-oxa-1,3-diazole; NBD-PE, N-(7-nitrobenz-2-oxa-1,3- diazol-4-yl)-1,2-dihexadecanyol-sn -glycero-3-phosphoethanolamine; rhodamine, 5(6)-carboxytetramethylrhodamine; Rh-PE, Lissa- mine TM rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phospho- ethanolamine, triethylammonium salt; SA, simulated annealing; SU, surface; TM, transmembrane. (Received 2 6 May 2004, revised 6 October 2004, accepted 13 October 2004) Eur. J. Biochem. 271, 4725–4736 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04436.x 1,2-dimy ristoyl-sn-glycero-3-phosphoglycerol (DMPG) were obtained from A vanti Polar L ipids (Alabaster, AL, USA). 7-Nitrobenz-2-oxa-1,3-diazole (NBD) and proteinase K were purchased from Sigma (St. Louis, MO, USA). 5(6)-Carboxytetramethylrhodamine (TAMRA), N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanyol- sn-glycero-3-phosphoethanolamine (NBD-PE) and Lissamine TM rhodamine B 1,2-dihexadecanoyl-sn-glycero- 3-phosphoethanolamine, triethylammonium salt (Rho-PE) were purchased from Molecular Probes, Inc. (Eugene, OR, USA). S DS and d 25 -SDS were acquired from Boeh- ringer Mannheim (Mannheim, Germany) and Cambridge Isotope (Andover, MA, USA), respectively. Solutions con- taining vesicles were p repared by solubilizing the lipids i n chloroform/methanol (4 : 1, v/v) mixture and drying the sample under n itrogen stream b efore dissolving i n buffer solution. Peptide/SDS mixtures a nd peptide/phospholipid mixtures were sonicated for 30–60 min before measure- ments. The internal fusion peptide Ac-GPTARIFASILAPG VAAAQALREIERLA-NH 2 (IFP–wt), residues 16–43 of the native envelope protein from the avian sarcoma and leukosis virus s ubtype A , a nd its P 13F ( IFP–F13), P 13V (IFP–V13) variants were assembled by Fmoc/t-Bu solid phase peptide synthesis as C-terminal carboxyamides on Fmoc-Rink amide Resin using a peptide syn thesizer (Pro- tein Technologies, Tucson, AZ, USA, model Rainin PS3) operated in the manual mode. The N-terminal free peptides were labelled with TAMRA following the standard Fmoc- amino a cid coupling protocol with a coupling t ime of 10 h. Labelling with NBD was achieved according t he procedure of Rapaport and Shai [ 13] with modifications as described in a recent s tudy [14]. Cleavage and purification of the peptide were as described previously [14,15]. The N-terminal peptide corresponding to residues 1–25 of HA2 (strain X31) of influenza virus, HA2[1–25], w as also synthesized [15] for comparison of structure and f unction. Circular dichroism experiments CD measurements were carried out on a Jasco 720 spectropolarimeter. E ach o f the peptides tested was incu- batedwithNaCl/P i or DMPC/DMPG (1 : 1) vesicular suspension, at pH 5.0 or 7 .4 to give a final concentration of 30 l M of peptide in NaCl/P i or of peptide/DMPC/DMPG (12 l M :0.8m M :0.8m M ). All samples were measured with a 1.0-mm path length cell, at 37 °C. The spectra were recorded from 260 to 190 nm at a scanning rate of 50 nm Æmin )1 with a time constant of 2 s, step resolution of 0.1 nm, and bandwidth of 1 nm. The final spectra were taken from the average of five s cans. The V ARSELEC program was used for the secondary structure prediction as described previously [16,17]. Fluorescence spectrometry All fluorescence experiments were performed on a Hitachi F-2500 Fluorescence Spectrometer at 37 °Cusinga1cm 2 semimicro quartz cuvette with stirrer. The response time was set at 0.08 s, slit bandwidth for excitation and emission was 10 nm. A scan rate of 300 nmÆmin )1 was used for the wavelength scans. Membrane binding and depth of immersion of the peptide probed by N-terminally labelled NBD The NBD-labelled peptide was used to m onitor the interaction between the peptide and lipid vesicles of DMPC/DMPG (1 : 1, molar ratio) with a suspension containing 0.06 l M and 300 l M of the NBD-labelled peptide and phospholipid, respectively, at pH 5.0 or 7.4 [18]. T he excitation and emission wavelengths w ere set at 467 and 530 nm, respectively, for time scan measure- ments. Spectra in the 500–650 nm range were collected in the wavelength scan experiments. The digestive enzyme, proteinase K (60 lgÆmL )1 in final concentration), was added to vesicles loaded with NBD-conjugated peptide to investigate the extent of protection from the enzyme action by the membrane on each of the peptides tested. For Co 2+ quenching experiments, the NBD-labelled peptide was added to a cuvette containing DMPC/DMPG vesicles at pH 5 or pH 7.4 and measurement was taken until the fluorescence signal attained a steady value. The final concentrations of peptide/DMPC/DMPG w ere 0.06 : 150 : 150 l M . A n i ncremental amount of CoCl 2 stock s olution (0.1 M ) was then injected into the cuvette to give final concentrations in the range of 0.02–2.0 m M . Corrections due to dilution were made to the observed fluorescence intensi- ties. T he data were analyzed using the Stern–Volmer equation: F 0 =F ¼ 1 þ K sv [Q] where F 0 and F are the intensity o f NBD fluorescence before and after addin g a given amount of CoCl 2 solution, respectively, [Q] is the concentration of the quencher and the slope K SV is the Stern–Volmer constant. Lipid mixing assay by fluorescence resonance energy transfer Membrane fusion assay used in t he study is based on the measurement of FRET from NBD to rhodamine [19]. Specifically, two lipid suspensions were prepared, one unlabelled ( DMPC/DMPG 2 50 : 2 50 l M )and one labelled (DMPC/DMPG/NBD-PE–Rho-PE 250 : 250:5:5l M ), using NaCl/P i buffer at n eutral or acidic pH. A 9 : 1 molar ratio o f unlab elled to l abelled liposomes (total volume 1 mL) was used in the assay; hence the final DMPC/DMPG to NBD-PE molar ratio is 1000 after the lipid mixing. Various aliquots of 1 m M peptide stock solution dissolved in dimethylsulfoxide (DMSO) were injected into the liposome mixture. As a control, 20 lL DMSO was used. As the fusion peptide is added to induce lipid mixing, the fluorescent probe is diluted by m ixing of t he unlabelled a nd labelled v esicles, resulting in r educed energy transfer efficiency and an increase in the fluorescen ce intensity of the ener gy donor, NBD-PE. T o monitor the NBD probe, the excitation and the e mission wavelengths were set at 467 nm and 530 nm, respectively. The fluorescence inte nsity after the a ddition of Triton X-100 (0.2% v/v) was referred to as 100%, respectively. 4726 S F. Cheng et al. (Eur. J. Biochem. 271) Ó FEBS 2004 Self-association tendency of IFP–wt by N-terminally labelled rhodamine fluorophore The rhodamine self-quenching experiments were carried out to examine the propensity of self-association o f the peptides in NaCl/P i and in t he vesicular su spension. Briefly, the rhodamine-labelled peptide (0.1 l M ) in a queous buffer at pH 5.0 or 7.4 was mixed with DMPC/DMPG (1 : 1, molar ratio) vesicles (lipid concentration 200 l M ). To monitor t he rhodamine probe, the excitation and emission wavelengths were set at 530 and 578 nm, r espectively. Digestion of the labelled peptides by proteinase K (50 lgÆmL )1 )leadsto disassembly of the membrane-associated oligomeric fusion peptides, resulting in dequenching of rhodamine fluores- cence. The 100% reference intensity was taken from the fluorescence measured in the peptide/lipid dispersion solu- bilized with 0.2% (v/v) Triton X-100. In the experiments on the composition variation of rhodamine-labelled peptide, the total (labelled plus unla- belled) peptide c oncentration w as kept cons tant at 0.01 l M while the fraction of labelled peptide, x , w as varied from 0.05 to 1 i n DMPC/DMPG (150 : 150 l M )vesicular suspension. The normalized emission intensity I x /x was plotted against x [20]. It is noted that intra-trimeric interaction is detected for x values near 1 since nearly all peptide molecules are labelled and quenching therefore arises predominantly from the close neighbours within the same trimer. In contrast, for low x values, t he probability of find ing a pair of labelled peptides is slim and h ence quenching arises mainly fr om labelled peptides in nearby t rimers. SDS/PAGE experiments to examine the oligomerization of IFP–wt, –F13 and –V13 in the membranous setting A PhastSystem TM (Pharmacia Biotech, Sweden) accom- panied with PhastGel Ò high density a nd PhastGel Ò SDS buffer strips was used for SDS/PAGE experiments, which is particularly suitable for molecules in the molecular mass range 1000–20000. Peptide samples were added to 6% SDS, 10% glycerol and 10 m M Tris-buffered solutions at pH 6.8 andheatedat55°C for 10 min. IFP analogues and markers (0.5 m M ; Pharmacia Biotech MW marker kit, code no. 80-1129-83) were loaded in a P hastGel Ò sample applicator 8/1 (code no. 18-1816-01). The running condition and staining method followed the procedures given in Phast- System handbook (ref. no. 80-1312-29 and 80-1312-30, respectively). Compositions of the buffer s ystem in the gel, buffer strips and solutions used for development can be found in the homepage http://www.apbiotech.com. Attenuated total reflectance-FTIR measurements Polarized ATR-FTIR spectra were recorded on a Boman DA8.3 spectrometer with a KBr beamsplitter and a liquid nitrogen-cooled MCT detector according to procedures described previously [20]. Each of the studied peptides (20 lg) and DMPC/DMPG (1 : 1, molar ratio) were mixed in chloroform/meth anol (1 : 1 , v /v) s olution and equili- brated with sodium pho sphate buffer at pH 5.0 or 7 .4 to give a final peptide/lipid molar ratio of 1 : 50. The sample was carefully spread on the germanium surface until solvent had e vaporated. T he ATR sample c overed with a home- made box was kept in full D 2 Ohydration(D 2 O/lipid ratio > 35) based on infrared absorbance ratio of D-O/C-H stretch peaks. Three hundred scans w ere collected at a resolution of 2cm )1 with triangular apodization and incoming r adiation was polarized with a g ermanium single diamond polarizer (Harrick, Ossining, NY, U SA). Before depositing sample, the 45° germanium ATR-plate (2 · 5 · 50 mm) was cleaned by a plasma cleaner (Harrick, Ossining, N Y, USA). Analysis of ATR-FTIR data was performed in accordance with a previous study [21]. NMR experiments NMR samples were p repared by dissolving the IF P–wt powder at 1 m M concentrationinH 2 O/D 2 O9:1(v/v) aqueous buffer or 100 m M d 25 -SDS micellar solution. Dilute H Cl or N aOH solution was used to adjust pH to 5.0. One- and two-dimensional 1 H N OESY and TOC SY NMR experiments were performed on a Bruker AMX-500 spectrometer at 298 K (in SDS m icellar solu tion) or 278 K (in aqueous solution), as described previously [22]. In deuteron/hydrogen (D/H) e xchange experiments, the pep- tide incorporated into SDS sample was lyophilized three times with pure H 2 O. D 2 O/H 2 O 9 : 1 (v/v) was added immediately before acquiring NMR data at 298 K a nd pH 5.0. To measure the effect of Mn 2+ ions on the relaxation behaviour o f IFP–wt protons, MnCl 2 dissolved in H 2 O was added to IFP–wt micellar solution to give a fi nal molar ratio of 0.696 (Mn 2+ /IFP–wt). Mn 2+ is an aqueous ionic probe which is excluded from t he apolar core of the micelle. Because the relaxation rate enhancement varies inversely with the distance between the proton a nd the probe, t he protons located more d eeply in the micellar interior will be less affected by the spin p robe. The fraction of attenuated backbone amide proton s ignal is taken as the fractional intensity difference in the cross-peaks of NH/ aH or side-chain protons of a given res idue obtained for the protonated and deuterium-exchanged peptide (for exchange experiments) or before and a fter introduction of Mn 2+ to d 25 -SDS micellar s olution (for relaxation enhancement measurements). Structure calculations Using d istance geometry (DG)/simulated annealing (SA) protocols of BIOSYM programs INSIGHTII , DISCOVER and NMRCHITECT (version 2000.1) from Accelrys Inc. ( San Diego, CA, U SA), 360 constraints were e mployed i n the structural computations (Table 1). The intermolecular NOE r estraints were classified semiquantitatively into three categories: strong (less than 2.6 A ˚ ), medium (2.6–3.6 A ˚ )or weak (3.6–4.6 A ˚ ). A range of 0.6–2.0 A ˚ was allowed to vary in the distance constraints. In the SA protocol, the temperature was raised to 1000 K in f our s teps followed by amoleculardynamicsrunfor30pstoallowmore conformational space to be explored. The system was subsequently annealed to 300 K in 10 steps for a total of 55 ps and minimized by the steepe st-descent and conju- gated-gradients m ethods before final refined structures were obtained. Ó FEBS 2004 Structural study of internal fusion peptide of ASLV (Eur. J. Biochem. 271) 4727 Results CD experiments on IFP–wt and -F13 indicate helix enhancement of the two peptides upon associating with lipid bilayer Figure 1 displays CD data on the two internal FP analogues in aqueous s olution and i n DMPC/DMPG (1 : 1) v esicular dispersions at pH 5.0 and 7.4 at 3 7°C. No sign ificant change in the s econdary structure o ccurs upon acidification fr om pH 7.4–5.0 for both analogues in DMPC/DMPG disper- sion; the only peculiarity is a dramatic increase in helicity with acidic pH for the F13 analogue in a queous solution. On the other hand, the helix content is increased when IFP– wt is transferred from aqueous to vesicular suspension, in analogy to the N-terminal FP such as that of gp41 [22] and of influenza HA2 [23], demonstrating the c onformational plasticity of the v iral fusion pepti des. H owever, the helix population in the membranous e nvironment is h igher for IFP–wt than for these two N-terminal FPs. The h igh helix content of F13 varian t in aqueous solution at acidic pH may reflect the c ritical effect of proline on the helical propensity of the fusion peptide sequence, but this structural effect diminished on as sociating with t he membrane. A s will be further demonstrated by NMR r esults, helix is induced in the N-terminal portion of the peptide in the membranous environment. Binding of IFP–wt to membrane bilayer is detected by NBD-labelled peptide To investigate the natur e of me mbrane interaction o f I FP–wt as manifested in the secondary structure change o f the peptide upon binding to the membrane (Fig. 1), we utilized IFP–wt with an N-terminally attached NBD which exhibits greatly increased fluorescence emission in a less polar environment. As illustrated in Fig. 2A, the fluorescence intensity increased several f old when the labelled peptide was transferred from aqueous buffer to vesicle dispersions, providing direct evidence that the peptide (or at least its N-terminal porti on) penetrates i nto t he membrane apolar interior. To further investigate t he insertion depth, Stern– Volmer constant K SV obtained from N BD quenching b y Co 2+ was utilized. The K SV was calculated from the linear part of Fig. 2B (in the range of [Co 2+ ] ¼ 0–0.3 m M ). The data reveal that acidification of vesicular dispersion results in deeper immersion of the peptide , at least at the N-terminus, as reflected by a smaller K SV . In the bottom panel of Fig. 2B, data from HA2(1–25) were used to compare the insertion depth between the N-terminal and internal fusion peptides. The N-terminal region of the N-terminal fusion peptide is seen to penetrate more deeply than that of IFP–wt. The result Table 1. C onstraints used for molecular simulation calculations on IFP–wt in sodium dod ecyl sulfate micelle and the deviations f rom the average stru ctures. Constraint type Total no. constraints No. constraints Constraints Chiral 30 Dihedral 26 Distance 304 Subtype Sequential 139 Medium i,i+2: 54 i,i+3: 55 i,i+4: 52 Long a 4 RMSD type Deviation range No. of deviation Distance 0.5–1.0 A ˚ 17 > 1.0 A ˚ 0 Dihedral > 5° 19 >10° 7 >15° 7 >20° 0 a d(i,j) constraints where j 3 i+5. Fig. 1. Far-UV CD spectra of ASLV IFP–wt and IFP–F13 in aqueous buffer and in DMPC/DMPG vesicle media at pH 5.0 (top panel) and 7.4 (bottompanel)at37°C. Helix content estimated from the ellipticity value a t 222 n m is en hanced considerably as IF P–wt was trans ferre d from aqu eous to vesicular solution at both pHs; this is true for IFP–F13 only at pH 7.4. The CD spectra of IFP–V13 are similar to those of IFP– F13 under the same conditions. The insets to the panels display the results of the secondary s tructure analysis with the VARSELEC program. 4728 S F. Cheng et al. (Eur. J. Biochem. 271) Ó FEBS 2004 will be discussed i n c onjunction with NMR data (Fig. 7) obtained in SDS micellar solution. Furthermore, greater insertion depth is observed for both fusion peptides at acidic pH than at neutral p H. This may h ave implications on the fusion pheno type of the viral fusion proteins since the fusion function of both e nvelope proteins is sensitive t o pH [24]. Fluorescence resonance energy transfer data demonstrate the lipid mixing activity of IFP–wt To investigate the fusogenicity of the peptide, NBD- and Rho-labelled PE w ere used a s the donor and acceptor, respectively, of fluorescence energy transfer. Dilution of the fluorescent-labelled PE loaded into v esicles by the unloaded vesicles via membrane fusion induced by the fusion peptide results in a reduction in the fluorescence energy transfer efficiency, hence d equenching of the donor fluorescence. As illustrated in Fig. 3, lipid mixing as de fined in Experimental procedures is plotted against peptide to lipid ratio i ndicates that the peptide is capable of promoting lipid mixing of vesicles consisting of DMPC and DMPG. Additionally, a peptide d erived from randomized sequence of IFP–wt (see Supplementary data) exhibited insignifi cant lipid mixing activity, demonstrating the specificity of the activity of the IFP–wt sequence. Inspection of Fig. 3 revealed that lipid mixing activity is higher for IFP–wt than the N-terminal fusion peptide, HA2[1–25], of influenza v irus at the same peptide-to-lipid ratio. The result is analogous to the data on the Sendai viru s observed by Peisajovich et al. [ 25] showing that the IFP has higher fusion activity than t he N-terminal FP. Self-quenching of rhodamine fluorescence indicates self-assembly of IFP–wt in the lipid bilayer To further examine the organization of the fusion peptide in the membrane, the peptide was l abelled with rhodamine [26]. Self-association of the molecules is monitored by the Fig. 2. Mem brane binding and insertion depth o f IFP–wt p robed by NBD fluorescen ce. (A)LipidbindingofNBD-IFP–wtatpH7.4and 37 °C. I ncreased intensity and the blue-shift of flu orescence of NBD attached to the N-terminus o f the peptide in dicate emb ed ding o f the peptide i n the apolar milieu of membrane bilayer. As a control, pro- teinase K digestion of the peptide disrupts membrane binding releasing bound NBD an d thus re du ces fluore scence of the fluorophore. (B) Stern–Volmer plot of cobalt quenching of NBD-IFP–wt to probe the immersion dept h in D MPC/DMPG vesicular suspension at pH 5.0 and 7.4. K SV values of HA2(1–25), the fusion peptide of influenza virus are compared to IFP–wt. The calculated K SV values (based on the data in the range of [Co 2+ ] ¼ 0–0.3 m M ) were sho wn in the inset. For both fusion peptides, K SV is smalle r a t lower pH, indicating deeper penet- ration than at neutral pH. At the s ame pH, larger K SV for IFP–wt shows shallower immersio n o f t he peptid e i n t he vesicl e t han t he N-terminal FP. Fig. 3. Lipid mixing induced by IFP–wt as probed b y FRET at 37 °C. NBD-andRho-labelledPEwereincorporatedinthevesiclestowhich wereaddedfusionpeptideandunlabelled lipid disper sion. Mixing rates are plotted against peptide-to-lipid ratios for I FP–wt and the N-terminal FP of influenza virus. Fusion activity exhibits strong pH de pendenc e for the IFP–wt. Lipid mixing rate is larger fo r IFP–wt than the i nfluenza fusion peptide at a given P/L-value . D equenc hing of the donor N BD b y dilution of the acceptor Rho resulting from p eptide-me diated membrane fusion is normalized with respect to the intensity obtained from lysis of vesicles w ith 0.2% Triton X-100. Ó FEBS 2004 Structural study of internal fusion peptide of ASLV (Eur. J. Biochem. 271) 4729 self-quenching of rhodamine fluorescence. The result shown in Fig. 4A for IFP–wt i n DMPC/DMPG vesicular suspen- sion indicates a moderate (approximately 40%) dequench- ing after addition of the d etergent (Triton X-100) to the suspension, suggestive of a loose a ssociation for the peptide in the membrane bilayer. Self-assembly can also b e analyzed by compositional variation of rhodamine-labelled p eptide, keeping the total concentration of labelled and u nlabelled peptides constant. In the experiments le ading to Figs 4B, 0.01 l M of total peptide was incorporated in DMPC/DMPG (150 : 150 l M ) vesicle dispersion for IFP–wt, with composition of the labelled peptide (x) varying from x ¼ 1–0.05. All three peptides exhibit c haracteristic of m ultimeric species in the lipid bilayer, as displayed b y self-quenching of rhodamine at x ¼ 1 compared to intensity at x ¼ 0.05 (for example, quenching efficiency in excess of 3 for IFP–wt) and the shape o f I x /x vs. x plot. Specifically, the initial slow rise of the l atter plot (from x ¼ 1) reflects that the self-association of the peptide is not tight ( on the scale of self-quenching distance of rhodamine,  15 A ˚ [17]). The deviation of the observed profiles from calculated ones based on homogen- eous clustering of various multimers ( N ¼ 1, 2, 4 and 8 ) indicates more than one mode of association of the FP molecules, involving m ore tightly packed oligomeric (such as trimer as in other class I fusion proteins) subunits interacting loosely with neighbouring oligomers. Thus, the sharp i ncrease in I x /x near the x ¼ 0 r egion shows dequenching of the probe and hence a tendency toward random distribution of t he peptide in t he membrane in the long distance range (> 30 A ˚ ), indicating no large scale aggregation occurs for the peptide. SDS/PAGE experiments suggest propensity of self-association of IFP–wt in the membrane-mimic environment Self-assembly of IFP–wt in the membranous environment canalsobediscernedbySDS/PAGEdatashowninFig.5, which also displays the results for IFP–F13 and IFP–V13. All peptide analogues exhibit diffuse bands and migrate roughly as dimeric species, with F13 and V13 variants forming oliogomers of slightly higher mass. The data are in qualitative agreement with the rhodamine self-quenching result for IFP–wt ( Fig. 4). All peptides exhibit little tendency of forming a large and tight molecular cluster. As SDS micelle is considered to be strongly disruptive o n Fig. 4. Self-a ssembly of the internal fusion peptide analogues i n associ- ation with DMPC/DMPG vesicles at pH 7.4 and 37 °C. (A) Relative intensity of Rho-labelled IFP–wt, –F13 and –V13 i n aqueous buffer (unfilled bar), DMPC/D MPG vesicle (grey bar) a nd vesicle t reated with proteinase K (black bar). The results indicate that IFP–wt has lower propensity of forming oligome r in the vesicular me dium than the other two varian ts and is probably monomeric in aq ueous solution as Rho self-quenc hing is le ss than in the presence of v esicles for the labelled peptide. (B) Normalized Rho emission intensity as a function of th e fraction o f labelled peptide as a probe for self-aggregation. As indicated in the plot, monome ric and dimeric species fo r th e p eptide are re presented by the h ypoth etical h or izontal and diagonal lines, respectively. The nearly unchanged I x /x in the h igh x regio n reflects a tight shorter range, probably intratrimeric, packing but the sharp rise in the low x region indicates a loose association for longer r ange inter- trimeric interaction. This interpretation is based on the fact that, at low x limit, th e probability of finding a labelled FP within a trimer for a g iven rhodamine p robe is low thus the result emphasizes inter-trimer interaction; the r everse is true in the x ¼ 1 limit. M oreover, the d evi- ation of t he experimental curves f rom those calculated by assuming a single species of a ssociation of N monomers indicates a m ulti-mode association for the peptide analogues in the membrane bilayer. The data therefore imply a heterogeneous distribution of FP molecules, which can be interpreted by a more close-packed trimers interacting loosely with a djacent trimers. Fig. 5. SDS/PAGE measurements on the molecular association for the three IFP analogues. In accord with the data o f Fig. 4A, IFP –wt has lower propen sity of forming high order oligomer th an t he o ther two analogues. 4730 S F. Cheng et al. (Eur. J. Biochem. 271) Ó FEBS 2004 the non–covalent interaction, the data of Fig. 4 provide indirect evidence for p ropensity of IFP–wt and analogues for self-assembly in the membranous medium. Helical structure and insertion angle of the membrane-associated fusion peptide as measured by FT-IR spectroscopy The secondary structure of peptides can also be quantitated by infrared spectroscopy. As shown i n F ig. 6 , the helical content calculated f rom t he band at 1655 cm )1 after deconvolution is 60% (Table 2), i n agreement with that observed in the CD data of the wild-type peptide in the vesicular s uspension at pH 7.4 (cf. Figure 1). The helix population i n IFP–wt is h igher, while the b-sheet content is lower, than that in the fusion peptide of influenza virus [20]. Slightly higher helix content was found for IFP–F13 (64%, Table 2), and IFP–V13 (66%, data not shown), suggesting helix structure i s not a sufficient determinant f or the f usion activity of the internal fusion peptide of ASLV. Secondary structure composition of the peptide varied little, if any, with pH between 5.0 and 7.4 (C.W. Wu and D.K. Chang, unpublished observation). The insertion angle of t he peptide helix deduced from the polarized ATR FT-IR result (Table 2) is  53° with respect to the membrane normal. Compared to N-terminal f usion peptides such as that of influenza virus [20], the result indicates that IFP–wt associates with the bilayer in a more shallow fashion. Still shallower insertion was obtained for the IFP–F13 variant. There is an insignificant change in insertion angle as pH varies from 5.0 to 7.4. Secondary structure change and insertion depth for membrane-bound fusion peptide can be determined on the residue level by NMR measurements The secondary structure and membrane insertion of the fusion peptide can be examined at the atomic level by NMR spectroscopy using a micelle as a model. Figure 7 A summarizes the NOE interactions from proton p airs with distance ¼ 5A ˚ in SDS micellar dispersion and in aqueous buffer solution at p H 5.0. T he d aN (i,i+3) and d aN (i,i+4) interactions, c haracteristic of helix structure, can be found in the residues 1–10 and residues downstream of Pro13 in SDS micelles. In accord with NOE data, helical segments, which Fig. 6. ATR-FTIR results on IFP–wt in DMPC/DMPG vesicles at pH 7 . 4. The top panel displays data from polarized FT-IR experi- ments; the ratio of th e parallel to perpe ndicular components relates to the angle of insertion of t he FP molecules into the bilayer. || and ^ represent parallel and perpendicular polarized light, respectively. The bottom panel displays the result of deconvolution of the IR absorption spectrum of the peptide f or analysis of the sec ondary struc ture. The dominant pe ak at 1655 cm )1 is attributed to the helical structure. The dottedandsolidtracesareexperimentalanddeconvoluteddata, respectively. Table 2. S econdary structure and helix orientation of IFP–wt and -F13 in DMPC/DMPG vesicular dispersion as deduced from ATR-FTIR data. a The peptide-to-lipid molar r atio was 1 : 50. b H values are angles between the helix axis and the bilayer normal. pH 5.0 pH 7.4 IFP–wt IFP–F13 IFP–wt IFP–F13 Secondary structure percentage a-Helix 57 ± 3 66 ± 3 60 ± 4 64 ± 3 Disorder 12 ± 2 11 ± 3 12 ± 3 11 ± 3 b-Sheet 24 ± 1 20 ± 2 22 ± 1 22 ± 1 b-Turn 7 ± 1 3 ± 2 6 ± 1 3 ± 1 Helix axis orientation Order parameter S amide I¢ 0.032 ± 0.02 )0.19 ± 0.03 0.095 ± 0.04 )0.14 ± 0.06 H 53° 63° 51° 60° Ó FEBS 2004 Structural study of internal fusion peptide of ASLV (Eur. J. Biochem. 271) 4731 are characterized by consecutive r esidues with spin–spin coupling constant 3 J aN ¼ 5 Hz, can be identified for t he residues 3–10 and 14–28 as shown i n Fig. 7B. Also plotted in Fig. 7B are 3 J aN data in aqueous buffer, indicating residues 16–21 are more helical while residues 6–10 have more b-strand character. C omparing 3 J aN values in aqueous buffer and in mice llar s uspension, it is clear that helix is induced in the N-terminal half f or the peptide in association with SDS micelle. Thus helix enhancement o f t he peptide on binding to the membrane shown in Fig. 1 is corroborated by the data of Fig. 7. The backbone amide deuterium/proton (D/H) exchange can be used to p robe the immersion of the peptide in SDS micelle with the notion that slower exchange correlates with deeper penetration into the micelle for the residue under consideration. Another method to gauge the insertion depth is a r eduction of resonance intensity resulting from r elax- ation enhancement by the aqueous spin probe, manganese, the extent of which is highly dependent on the Mn 2+ – proton distance [27]. The results o f these two experiments as summarized in Fig. 7C are consistent in that the region encompassing Pro13 is near the micellar headgroup region and the residues 16–21 penetrate more d eeply into the micellar interior than o ther regions of the peptide. Accord- ing to Fig. 7C, the backbone of Arg22 resides near the apolar–headgroup interface of the micelle; its side chain is likely extended to t he headgroup region of the micelle resulting in neutralization o f the positive ch arge by the sulfate group. Fig. 7. Sec ondary structure an d t opology of IFP–wt in SDS micelles determined by NMR spectroscopy. (A) NOE interactions of IFP–wt in aqueous buffer ( top) an d i n a ssociation w ith SD S mic elles ( bottom). (Upper) Folded structure can be discerned in the region 10–18 and 20– 26. (Lower) Helical segments can b e observed i n 1–11 a nd 14–28 by contiguous (i,i +3) interactions. The absence of (i,i +3) cross-peaks between L eu 11 and Gly14 is consistent with a distorted h elix for the region aro und Pro13. (B) 1 H sp in–spin coupling constants ( 3 J aN )for the residues of I FP–wt me asured i n aqu eous solutio n and S DS mice llar suspension. Residues of the helical and b-sheet structure are charac- terized b y values smaller than 5 Hz and greater than 8 Hz, respect- ively. The tran sformation into helix of the N-terminal regions is obviousasIFP–wtistransferredfromaqueoustomicellarmedium. (C) Attenuation o f the intensity o f NH -aHcross-peaksinD/H exchange and Mn 2+ relaxation enhancement experiments. The standard error in computing signal attenuation is 5%. Mo re exposed backbone amide proton results in faster ex cha nge between deuterium and proton and hence smaller cro ss-peak reten tion. The relaxation enhancement o f the backbone proto ns is inversely proportional to the sixth p ower of Mn 2+ -proton distance; larger signal retention therefore represents greater dep th of insertion of the amino a cid residue into the micelle. Taken together, the two sets of data indicate t hat the stretch around Pro13 is closer to the surface and the C-terminal half penetrates more deeply than t he N-terminal half of IFP–wt. Fig. 8. NH- aH and NH -side chain proton region of 1 HNOESYspec- trum of IFP–wt in the aqueous medium. Interactions between the resi- dues around Pro13 used to determine the type II b-turn are indicated. Particularly not eworthy are the cross-peaks attributed to bHofAla12 and NH o f Val15, and aH of P ro13 and NH o f Val15. 4732 S F. Cheng et al. (Eur. J. Biochem. 271) Ó FEBS 2004 Conformational alteration of IFP–wt occurs when the peptide is transferred from aqueous solution to SDS micellar dispersion, particularly in the region near Pro13 NMR data of IFP–wt suggest an overall lower helicity in aqueous solution compared to that in SDS micellar solution. Because of the helix-disruptive property of proline, we further e xamined the structure o f the region adjacent t o Pro13 of IFP–wt. Several lines of evidence from NMR data shown in Fig. 8 suggest that Pro13-Gly14 essentially form a type II b-turn in aqueous solution, while a slightly kinked helix is deduced in the presence of micelles. First, in aqueous medium coupling constant 3 J aN for Gly14 is  6 Hz, closed to 5 H z in a type II b-turn. Second, the r esult that d aN (Pro13,Gly14) is stronger than d aN (Gly14,Gly14) indi- cates t hat a type II instead of t ype 1 turn is adopted for the Ala12-Pro13-Gly14-Val15 stretch. Third, the relationship of interproton distance a lso provides evidence of the claimed turn structure. Thus d NN (Gly14,Val15) is short for the strong inten sity o f the cross-peak between the a mide protons of the two residues; d aN (Gly14,Val15) is shorter than in traresidue d aN (Val15,Val15); a prominent d bN (Ala12,Val15) peak is observed but not other d bN (i,i +3) for i ¼ Ser9, Ile10 and Leu11, suggesting a b-turn exists in the segment Ala12-Pro13-Gly14-Val15. A turn-to-helix conformational change is a lso manifested in the upfield shift of Val15 C a H from 4 .05 to 3 .85 p.p.m. as SDS micelles are added to the medium although the helix is distorted near Pro13 in the presence of the micelle. Another r esult in support of the contention that Val15 is the fourth residue of a t urn is a fforded by the small t emperature coefficient of NH of Val15, namely 2.5 · 10 )3 p.p.m.Æ°C )1 (compared to 8.5 · 10 )3 p.p.m.Æ°C )1 for G ly14 NH) in aqueous solution, suggesting that its N H p articipates i n hydrogen bonding. The latter notion is also corroborated by a distinct d aN (Pro13,Val15) cross-peak. A kink at Pro13 is visualized by the structural calculation based on the proton NOE and coupling constant constraints To obtain three-dimensional structure, we employed INSIGHTII/DISCOVER and NMRCHITECT using constraints derived from NOE and 3 J aN . Figure 9 illustrates the superposition of 20 structures (PDB ID: 1XNL). Detailed structural statistics are tabulated in Table 1 . Note that the helices have a kink of  20° at Pro13, consistent with the effect of th e proline on the helix summarized by Barlo w and Thornton [28]. Discussion We have studied the s tructure of the IFP of ASLV-A and its membrane interactions to examine the difference in the biophysical property b etween the N-terminal and internal FPs, in the hope of improving our understanding of the role of fusion peptides in the fusion e vent. Structure and membrane interaction of IFP–wt Binding of IFP–wt to lipid vesicles as demonstr ated in Fig. 2 supports the v iew that t he IFP o f A SLV is e ither responsible for or contributes to the binding of the virus to liposomes after exposure o f the fusion peptide in response to the conformational change triggered by interaction with the specific receptor [8]. As is the case for many IFPs, a proline is located near the centre of the FP sequence. Both CD and NMR data indicate a higher helix content than t he N-terminal FP (e.g. influenza H A2 and HIV-1 gp41) in the membrane-mimic environment. Figures 1, 7 and 8 also demonstrate b–a transformation when th e peptide is transferred from aque- ous to micellar medium; more specifically, NMR results indicate that the helical structure is in duced mainly in the region N-terminal to proline and there is a slight kink in the helix at Pro13. As shown i n Fig. 7C, the region surrounding Pro13 r esides closest to t he micellar surface. A kinked h elix structure has also been deduced from an NMR study on the fusion peptide of HA2 in dodecylphosphocholine micellar solution [29], indicating that it is therefore probably a common f eature for the class I viral fusion peptides embedded i n the membrane and is involved in destabilizing the membrane during the fusion process. The notion is supported by a mutational study on ASLV fusion protein in which the viral infectivity was found largely unaffected by the substitution of Pro13 with a residue thought to retain the bending or flexibility of the central region of IFP [30]. Except for t he moderate distor tion on the helical structure, Pro13 does not significantly reduce helicity of the wt-peptide in lipid a s observed from the comparison of CD data (Fig. 1 ) between the p eptide and i ts F13 variant. The conformational switch for the stretch near Pro13 from a type I I b-turn to a kinked h elix as the f usion peptide is transferred from aqueous to SDS micellar medium underscores our previous contention r egarding t he Fig. 9. Supe rposition of 2 0 structures of IFP–wt calculated with t he constraints derived from NMR data performed in SDS micelles. Akink caused by Pro13 s hows that th e a mino acid may have a structural effect on helix in the membrane-mimic medium s imilar to t hat in a queo us solution. Ó FEBS 2004 Structural study of internal fusion peptide of ASLV (Eur. J. Biochem. 271) 4733 N-terminal fusion peptide that the structural plasticity is germane to its fusogenicity and a proper balance between helix and b-sheet structure i s important for fusion activity. It is thus likely that both internal and N-terminal fusion peptides destabilize the target membrane in a s imilar fashion, n otwithstanding a shallower penetration of the IFP into the membrane. It is noteworthy that substitution of Pro13 by a residue with intermediate hydrophobicity (Thr for instance) resulted in an MLV-pseudotyped virus with h igher i nfectivity [8]; the activity, moreover, did not correlate with the predicted secondary structure propensity of the substituted amino acid. The result is in line with our finding that Pro13 is located in the interfacial region having transitional polarity. Introduction of a residue with extreme polarity may perturb the topology o f the fusion peptide and its interaction with the membrane bilayer. It is of interest to note the ph enotype of H IV-2 gp41 mutants [31]: t he amino acid substitution of the fusion peptide sequence that increases hydrophobicity of the region would enhance syncytium formation, while mutation that increases charge or polarity of the fusion peptide domain reduces syncytium-inducing c apacity of t he virus. Coupled with our previous finding of a deeper insertion of t he fusion peptide of HIV-1 gp41 into model membrane [22], the result of Steffy et al. [31] c orroborates the assertion that Pro13 of IFP–wt re sides near the interfacial region of the bilayer. The orientation and i nsertion depth w ere probed b y FTIR and NMR techniques. The angle between the helix axis and b ilayer surface deduced from FTIR d ata is  37° for IFP–wt ( 27° for IFP–F13) but  50° for HA2-FP [20]. Hence the IFP–wt inserts into bilayer less steeply than the N-terminal fusion peptide of i nfluenza virus. The insertion depth obtained from NMR data (D/H exchange and Mn 2+ probe) in the SDS micelle suspension reveals that the N-terminal portion of the N-terminal fusion peptide [15] is embedded mo re deeply than the C-terminal segment whereas the r everse is true for IFP–wt. Moreover, the estimated angle of insertion from the relaxation enhancement measurements is flatter and closer to the micelle surface for IFP–wt. This suggests th at IFP has a shallower i nsertion into the membrane. It also follows that penetration into both leaflets of the membrane bilayer is not required for the IFP to exert its f unction. The r esult is reasonable a s the charged and polar residues flanking the IFP sequence would p revent the FP from immersing too d eeply into the apolar core of the membrane. Our data are also compatible with the observation that truncation of t he membrane-spanning region of HA2 to the extent that the resulting segment cannot cross t he bilayer l ed to hemi- fusion but not complete fusion [32] and with the report that lipid-anchored influenza haemagglutinin promotes hemifusion [33]; both imply that the complete fusion cannot be supported b y t he fusion peptide domain alone and the transmembrane segment is indispensable. The deeper penetration at pH 5.0 than at pH 7.4 for both HA2 and ASLV fusion peptides as revealed by Fig. 2B may be r elevant i n t he pH dependence of fusion a ctivity for both v iruses; it may be that further perturbation of membrane structure with deeper insertion would promote progress to later s tages of fusion reaction, for example deeper penetration into membrane at acidic pH may facilitate p rogress from h emifusion achieved by ASLV at neutral p H t o pore formation [34]. Comparison o f t he orientation of IFP–wt and IFP–F13 in association with phospholipid bilayer (Table 2) also suggests that a small angle of insertion into the membrane may exist for the internal fusion peptide [35] to destabilize the membrane. An oblique insertion of fusion p eptide of SIV gp32 and influenza HA2 has been deduced by Martin et al.[36] and by Lu ¨ nerberg et al. [37] from FT-IR measurements. Lipid mixing activity of IFP–wt and its relationship to the secondary structure Lipid m ixing measurements on the internal and N-terminal fusion peptides indicate that the IFP has higher fusion activity than N-terminal fusion peptide of influenza v irus (Fig. 3 ). Both types of fusion peptides insert into the membrane bilayer obliquely and possess substantial regular structures (a-helix and b-sheet) in t he membranous medium, in supp ort of the idea that balanced helix and b-sh eet structures are necessary for fusion. Indeed, higher helix content alone does not correlate with fusogenicity as can be seen from comparison of helical structure between IFP–wt and IFP–F13. Organization of IFP–wt in model membranes An apparently dimeric band can be detected in the S DS/ PAGE measurement for IFP–wt. A species of slightly higher apparent molecular mass i s observed for each of the less fusogenic analogues IFP–F13 and IFP–V13, suggesting that oligomerization of fusion peptide m olecules i n t he membrane is not sufficient for their fusion activity. This i s i n contrast with the oligomerization propensity observed for HA2-FP and its mutants [17]. Both the simple rhodamine self-quenching and the rhodamine-labelled peptide compo- sition variation experiments (Fig. 4B) c learly demonstrate self-assembly for the membrane-bound peptides; the latter further provided e vidence o f m ultimodal s elf-assembly of the p eptide, a s deduced, in particular, from a steeper rise in I x /x in the region near x ¼ 0. The oligomeric structure for the ectodomain of the envelope glycoprotein of ASLV has also been inferred from sucrose gradient sedimentation experiments [38]. The results of Fig. 4 lead t o t he view that loose association of the IFP molecules in the membranous environment is r elevant t o the lipid mixing activity. In analogy to t he proposed multi-modal association, the clustering of trimeric envelope proteins on the surface of HIV virion has been elegantly shown by Zhu et al. [39] in their e lectron microscopic s tudy. Our data suggest t hat IFP contributes to the self-assembly of the envelope glycoprotein of ASLV bound to membrane. Effects of pH on the secondary structure and membrane interaction of IFP–wt Based o n studies on the molecular clustering of EnvA fusion protein of ASLV i n response to the receptor binding and low pH, Matsuyama et al. [24] proposed a fusion mecha- nism for the virus. Specifically, low pH promotes proceeding of fusion from outer monolayer mixing to complete fusion. 4734 S F. Cheng et al. (Eur. J. Biochem. 271) Ó FEBS 2004 [...]... between the internal and N-terminal fusion peptides in the membranous environment are: higher helix and lower b-sheet contents, shallower insertion for the internal fusion peptide More study on how the internal fusion peptide segment along with the heptad repeat regions and transmembrane anchor promotes membrane fusion would be very useful in the antiviral strategy Comparison of the membrane- fusion. .. study of internal fusion peptide of ASLV (Eur J Biochem 271) 4735 The result of Fig 3 suggests that the fusion activity of IFP– wt is higher while Fig 2 shows that the insertion depth is deeper at low pH Taken together, these data and the model of Matsuyama et al [24] are suggestive of the idea that deeper membrane insertion induced by acidic pH facilitates fusion of both inner and outer leaflets of fusing... gp41 fusion peptide [12] Furthermore, the core structure of the class I fusion proteins, which also undergoes structural rearrangement on transition to the fusogenic state, is predominantly helical in both pre- and postfusion states We have previously deduced from our NMR and fluorescence data that the fusion peptides of gp41 of HIV-1 and HA2 do not span both leaflets of the membrane bilayer [15,22] The. .. of fusing membranes Comparison between class I and II fusion proteins The crystal structures of soluble domain of two class II fusion proteins in association with detergent have been reported recently [10,11] The secondary structure of both proteins is mainly b-sheet in the putative pre- and postfusion forms The region corresponding to the fusion peptide of class I fusion protein exists in the membranous... membranous environment The present work represents an extensive biophysical study on the internal fusion peptide of a prototypic class I viral fusion protein We have observed similarities between the internal and N-terminal fusion peptides, namely, substantial helix enhancement on binding to membrane, loose self-assembly and that the fusion peptides do not traverse both leaflets of the membrane bilayer Some... IFP of ASLV-A is shown to penetrate more shallowly into the membrane than the N-terminal FP, in analogy to the IFP of class II fusion proteins [10,11] In conjunction with the model proposed for the class II fusion proteins, it is concluded that all the viral FPs and fusion loops examined thus far insert into the outer leaflet of the membrane Our result of variation of fluorescence quenching with the. .. compatible with the result on the interactions of soluble fusion protein fragments of Semliki Forest virus with membrane [10] Thus the insensitivity of the normalized intensity to variation in the labelled composition at the high fraction regime corresponds to intra-trimer interaction, whereas the steep intensity increase in the low label fraction regime reflects inter-trimeric interaction between the peptide. .. Conformational change and protein–protein interactions of the fusion protein of Semliki Forest virus Nature 427, 320–325 11 Modis, Y., Ogata, S., Clements, D & Harrison, S.C (2004) Structure of the dengue virus envelope protein after membrane fusion Nature 427, 313–319 12 Chang, D.K., Chien, W.J & Cheng, S.F (1997) The FLG motif in the N-terminal region of glycoprotein 41 of human immunodeficiency virus type 1... mature avian leukosis virus subgroup a envelope glycoprotein is metastable, and refolding induced by the synergistic effects of receptor binding and low pH is coupled to infection J Virol 78, 1403–1410 6 Hernadez, L.D & White, J.M (1998) Mutational analysis of the candidate internal fusion peptide of the avian leukosis and sarcoma virus subgroup A envelope glycoprotein J Virol 72, 3259– 3267 7 Boerkoel,... found that the proton chemical shift and 3JaN coupling constant values are close for the residues within the 37-mer peptide overlapping with IFP–wt Furthermore, the longer peptide exhibits lipid mixing activity and secondary structure similar to IFP–wt (see supplementary data) These results suggest that the conformation of the 28-mer IFP–wt is similar to the 37-mer cysteine-containing peptide in the membranous . in the hope of improving our understanding of the role of fusion peptides in the fusion e vent. Structure and membrane interaction of IFP–wt Binding of. Structure and membrane interaction of the internal fusion peptide of avian sarcoma leukosis virus Shu-Fang Cheng, Cheng-Wei Wu, Eric Assen B Kantchev and