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• W2006756069 abstract "The structural and antigenic properties of a peptide (“CRK”) derived from the V3 loop of HIV-1 gp120 protein were studied using NMR and SPR techniques. The sequence of CRK corresponds to the central portion of the V3 loop containing the highly conserved “GPGR” residue sequence. Although the biological significance of this conserved sequence is unknown, the adoption of conserved secondary structure (type II β-turn) in this region has been proposed. The tendency of CRK (while free or conjugated to protein), to adopt such structure and the influence of such structure upon CRK antigenicity were investigated by NMR and SPR, respectively. Regardless of conjugation, CRK is conformationally averaged in solution but a weak tendency of the CRK “GPGR” residues to adopt a β-turn conformation was observed after conjugation. The influence of GPGR structure upon CRK antigenicity was investigated by measuring the affinities of two cognate antibodies: “5023A” and “5025A,” for CRK, protein-conjugated CRK and gp120 protein. Each antibody bound to all the antigens with nearly the same affinity. From these data, it appears that: (a) antibody binding most likely involves an induced fit of the peptide and (b) the gp120 V3 loop is probably conformationally heterogeneous. Since 5023A and 5025A are HIV-1 neutralizing antibodies, neutralization in these cases appears to be independent of adopted GPGR β-turn structure. The structural and antigenic properties of a peptide (“CRK”) derived from the V3 loop of HIV-1 gp120 protein were studied using NMR and SPR techniques. The sequence of CRK corresponds to the central portion of the V3 loop containing the highly conserved “GPGR” residue sequence. Although the biological significance of this conserved sequence is unknown, the adoption of conserved secondary structure (type II β-turn) in this region has been proposed. The tendency of CRK (while free or conjugated to protein), to adopt such structure and the influence of such structure upon CRK antigenicity were investigated by NMR and SPR, respectively. Regardless of conjugation, CRK is conformationally averaged in solution but a weak tendency of the CRK “GPGR” residues to adopt a β-turn conformation was observed after conjugation. The influence of GPGR structure upon CRK antigenicity was investigated by measuring the affinities of two cognate antibodies: “5023A” and “5025A,” for CRK, protein-conjugated CRK and gp120 protein. Each antibody bound to all the antigens with nearly the same affinity. From these data, it appears that: (a) antibody binding most likely involves an induced fit of the peptide and (b) the gp120 V3 loop is probably conformationally heterogeneous. Since 5023A and 5025A are HIV-1 neutralizing antibodies, neutralization in these cases appears to be independent of adopted GPGR β-turn structure. principal neutralizing determinant human immunodeficiency virus-1 surface plasmon resonance fast protein liquid chromatography bovine pancreatic trypsin inhibitor double quantum spectroscopy double quantum filtered correlated spectroscopy 3-(trimethylsilyl)propanesulfonic acid antibody antigen-binding fragment fast atom bombardment nuclear Overhauser effect nuclear Overhauser enhancement spectroscopy o-methylisourea N-succinimidyl-3-(2-pyridyldithio)propionate total correlated spectroscopy glycoprotein resonance unit The principal neutralizing determinant (PND)1 of HIV-1 has been mapped to the third hypervariable (V3) loop of the HIV-1 envelope protein, gp120 (1Javaherian J. Langlois A.J. McDanal C. Ross K.L. Eckler L.I. Jellis C.L. Profy A.T. Rusche J.R. Bolognesi D.P. Putney S.D. Matthews T.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6768-6772Crossref PubMed Scopus (567) Google Scholar). PND-derived peptides are used as immunogens to elicit antibodies that possess HIV-1 virus neutralization capabilities (2Palker T.J. Clark M.E. Langlois A.J. Matthews T.J. Weinhold K.J. Randall R.R. Bolognesi D.P. Haynes B.F. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1932-1936Crossref PubMed Scopus (443) Google Scholar, 3Goudsmit J. Debouck C. Meloen R.H. Smit L. Bakker M. Asher D.M. Wolff A.V. Gibbs Jr., C.J. Gajdusek D.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4478-4482Crossref PubMed Scopus (410) Google Scholar). The binding of neutralizing antibodies blocks virus entry into the host cell but does not prevent binding of HIV-1 to its primary cell receptor protein, CD4 (4Wu L. Gerard N.P. Wyatt R. Choe H. Parolin C. Ruffin N. Borsetti A. Cardoso A.A. Desjardin E. Newman W. Gerard C. Sodroski J. Nature. 1996; 384: 179-183Crossref PubMed Scopus (1081) Google Scholar, 5Trkola A. Dragic T. Arthos J. Binley J.M. Olson W.C. Allaway G.P. Cheng Mayer C. Robinson J. Maddon P.J. Moore J.P. Nature. 1996; 384: 184-187Crossref PubMed Scopus (959) Google Scholar). Although the V3 loop represents an important target for the development of vaccines against AIDS, its high sequence variability also makes it a very problematic one (6LaRosa G.J. Davide J.P. Weinhold K. Waterbury J.A. Profy A.T. Lewis J.A. Langlois A.J. Dreesman G.R. Boswell R.N. Shadduck P. Holley H. Karplus M. Bolognesi D.P. Matthews T.J. Emini E.A. Putney S.D. Science. 1990; 249: 932-935Crossref PubMed Scopus (578) Google Scholar). Nonetheless, the occurrence of the highly conserved residue sequence, “GPGR,” at the tip of the V3 loop has raised the possibility that these residues make up a conserved secondary structural element in gp120. The function of such a structural element, if one exists, is presently unknown, however. The precise predicted secondary structure adopted by the GPGR residues is a type II β-turn (6LaRosa G.J. Davide J.P. Weinhold K. Waterbury J.A. Profy A.T. Lewis J.A. Langlois A.J. Dreesman G.R. Boswell R.N. Shadduck P. Holley H. Karplus M. Bolognesi D.P. Matthews T.J. Emini E.A. Putney S.D. Science. 1990; 249: 932-935Crossref PubMed Scopus (578) Google Scholar, 7Hansen J.E. Lund O. Nielsen J.O. Brunak S. Hansen J.E.S. Proteins Struct. Funct. Genet. 1996; 25: 1-11Crossref PubMed Scopus (32) Google Scholar). In order to determine whether the conserved GPGR sequence confers certain secondary structural tendencies upon this region of the V3 loop, numerous NMR studies were conducted upon a variety of V3 loop-derived peptides (8–21). These peptides were shown to have only low density populations of folded structure and to be conformationally averaged in solution. Despite the report of a “core” gp120 protein complex crystal structure, this complex was prepared using a form of gp120 which lacked most of the hypervariable loops including V3 (22Kwong P.D. Wyatt R. Robinson J. Sweet R.W. Sodroski J. Hendrickson W.A. Nature. 1998; 393: 648-659Crossref PubMed Scopus (2520) Google Scholar). Information regarding the actual three-dimensional structure of the V3 loop in native gp120 is therefore still unavailable. Due to the linear and flexible nature of V3 loop peptides in solution, a variety of methods have been employed to induce greater and presumably more “native” structure in previously studied V3 loop peptides. These methods included aminoisobutyric acid substitution (10Ghiara J.B. Ferguson D.C. Satterthwaite A.C. Dyson H.J. Wilson I.A. J. Mol. Biol. 1997; 266: 31-39Crossref PubMed Scopus (77) Google Scholar), insertion into a viral coat protein (23Jelinek R. Terry T.D. Gesell J.J. Malik P. Perham R.N. Opella S.J. J. Mol. Biol. 1997; 266: 649-655Crossref PubMed Scopus (49) Google Scholar), glycosylation (12Huang X. Smith M.C. Berzofsky J.A. Barchi Jr., J.J. FEBS Lett. 1996; 393: 280-286Crossref PubMed Scopus (28) Google Scholar, 13Huang X. Barchi Jr., J.J. Lung F.T. Roller P.P. Nara P.L. Muschik J. Garrity R.R. Biochemistry. 1997; 36: 10846-10856Crossref PubMed Scopus (90) Google Scholar, 14Markert R.L.M. Ruppach H. Gehring S. Dietrich U. Mierke D.F. Kock M. Rubsamen-Waigmann H. Griesinger C. Eur. J. Biochem. 1996; 237: 188-204Crossref PubMed Scopus (21) Google Scholar), attachment to resin beads (24Jelinek R. Valente A.P. Valentine K.G. Opella S.J. J. Mag. Reson. 1997; 125: 185-187Crossref PubMed Scopus (51) Google Scholar), cyclization of the peptide (15Chandrasekhar K. Profy A.T. Dyson H.J. Biochemistry. 1991; 30: 9187-9194Crossref PubMed Scopus (161) Google Scholar), and trifluoroethanol addition (14Markert R.L.M. Ruppach H. Gehring S. Dietrich U. Mierke D.F. Kock M. Rubsamen-Waigmann H. Griesinger C. Eur. J. Biochem. 1996; 237: 188-204Crossref PubMed Scopus (21) Google Scholar, 15Chandrasekhar K. Profy A.T. Dyson H.J. Biochemistry. 1991; 30: 9187-9194Crossref PubMed Scopus (161) Google Scholar, 16Zvi A. Hiller R. Anglister J. Biochemistry. 1992; 31: 6972-6979Crossref PubMed Scopus (67) Google Scholar, 17Vranken W.F. Budesinsky M. Martins J.C.K. Boulez K. Gras-Masse H. Borremans F.A.M. Eur. J. Biochem. 1996; 236: 100-108Crossref PubMed Scopus (35) Google Scholar, 18Catasti P. Fontenot J.D. Bradbury E.M. Gupta G. J. Biol. Chem. 1995; 270: 2224-2232Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 19Catasti P. Bradbury E.M. Gupta G. J. Biol. Chem. 1996; 271: 8236-8242Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). One particular method used previously to induce structure in short, linear peptides involves covalent attachment (or “conjugation”) of the peptide to BPTI protein. Using this method, a nine-residue peptide derived from hemagglutinin (25Dyson H.J. Cross K.J. Houghten R.A. Wilson I.A. Wright P.E. Lerner R.A. Nature. 1985; 318: 480-483Crossref PubMed Scopus (226) Google Scholar) was shown to significantly affect the solution structure of this peptide after its conjugation to BPTI (26Dyson H.J. Zegers N.D. Boersma W.J.A. Claassen E. Immunological Recognition of Peptides in Medicine and Biology. CRC Press, Boca Raton, FL1995: 133-145Google Scholar). Due to the well characterized NMR properties of BPTI protein (27Wagner G. Braun W. Havel T.F. Schaumann T. Go N. Wüthrich K. J. Mol. Biol. 1987; 196: 611-639Crossref PubMed Scopus (636) Google Scholar, 28Beeser S.A. Oas T.G. Goldenberg D.P. J. Mol. Biol. 1998; 284: 1581-1596Crossref PubMed Scopus (35) Google Scholar, 29Balasubramanian S. Nirmala R. Beveridge D.L. Bolton P.H. J. Magn. Res. B. 1994; 104: 240-249Crossref PubMed Scopus (16) Google Scholar, 30Wagner G. Wüthrich K. J. Mol. Biol. 1982; 155: 347-366Crossref PubMed Scopus (538) Google Scholar, 31Berndt K.D. Guntert P. Orbons L.P.M. Wüthrich K. J. Magn. Res. 1992; 227: 757-775Google Scholar, 32Nirmala N.R. Wagner R. J. Am. Chem. Soc. 1988; 110: 7557-7558Crossref Scopus (170) Google Scholar) and the fact that no modification of the peptide or solvent conditions are required, we chose to employ this method in an attempt to induce greater CRK peptide structure for these solution NMR studies. We began NMR and surface plasmon resonance (SPR) studies of a PND peptide in order to investigate the structural propensities of this peptide (while free and conjugated to BPTI), and their potential importance to the binding of this peptide by HIV-1 neutralizing antibodies. The PND peptide of interest, “RK,” has the following sequence: Arg1-Ile2-Gln3-Arg4-Gly5-Pro6-Gly7-Arg8-Ala9-Phe10-Val11-Thr12-Ile13-Gly14-Lys15. This sequence, corresponding to residues 308–322 of the gp120 envelope protein of HIV-1 (using the gp120 numbering scheme for strain IIIB, Ref. 33Durda P.J. Bacheler L. Clapham P. Jenoski A.M. Leece B. Matthews T.J. McKnight A. Pomerantz R. Rayner M. Weinhold K.J. AIDS Res. 1990; 6: 1115-1123Google Scholar), comprises the tip of the gp120 V3 loop and represents the center of the HIV-1 PND. The two antibodies studied, “5023A” and “5025A” were both raised against RK and both exhibit HIV-1 virus neutralization as well as cell fusion inhibition capabilities in vitro (33Durda P.J. Bacheler L. Clapham P. Jenoski A.M. Leece B. Matthews T.J. McKnight A. Pomerantz R. Rayner M. Weinhold K.J. AIDS Res. 1990; 6: 1115-1123Google Scholar). Since a cysteinylated peptide was needed for the protein conjugation study, the free and BPTI-conjugated form of an N-terminal cysteinylated form of RK (“CRK”) were actually studied and compared by NMR techniques. To determine the overall importance of folded CRK structure to its binding by its cognate antibodies, 5023A and 5025A, SPR techniques were used to measure the binding of these antibodies to various forms of the CRK antigen- free CRK peptide, BPTI-conjugated CRK peptide, and intact gp120. Based upon the NMR and SPR data presented in this study, the relationship between peptide antigenic structure versusantibody binding preference (for this PND peptide and these antibodies) is then discussed. A N-terminal cysteinylated form of peptide RK known as CRK, Cys−1-Arg1-Ile2-Gln3-Arg4-Gly5-Pro6-Gly7-Arg8-Ala9-Phe10-Val11-Thr12-Ile13-Gly14-Lys15, was synthesized and studied. The cysteine residue was required for conjugation of RK peptide to BPTI protein via a heterobifunctional chemical linker. This 16-residue peptide was synthesized using a Rainin Automated PS-3 Peptide Synthesizer and Fmoc chemistry. The crude peptide was purified using a Rainin HPXL HPLC System equipped with a Rainin Dynamax-300A reverse phase column with acetonitrile as the elution solvent. The peptide composition was verified by amino acid analysis and its molecular weight by FAB mass spectroscopy (Multiple Peptide Systems, San Diego, California). The peptide sequence and its purity (>95%) were evaluated by two-dimensional NMR techniques. The modification of BPTI and its coupling to CRK peptide was accomplished according to the method developed by Ebina et al. (34Ebina S. Lerner R.A. Wright P.E. J. Mol. Biol. 1989; 264: 7882-7888Google Scholar) with modifications. BPTI was modified using OMIU and then covalently linked to the chemical linker, SPDP, before peptide coupling. The modification of BPTI proceeds by conversion of its lysine residues to homoarginines. As a result, the N-terminal amino group remains as the only free amino group available for SPDP coupling. The cysteinyl side chain of the peptide is covalently attached to this linker. The protein was modified usingo-methylisourea and then purified using a Amersham Pharmacia Biotech LKB FPLC system and a Mono-S ion exchange column. A NaCl salt gradient in a 20 mm glycine, pH 10.5, buffer was used to purify the modified protein. The main protein fraction, which eluted at NaCl concentrations greater than 0.54 m, was collected. This fraction was then dialyzed extensively against deionized, distilled water and subsequently lyophilized. The molecular weight of the OMIU-modified BPTI (OMIU-BPTI) was determined via matrix-assisted laser desorption/ionization-time of flight mass spectroscopy to be 6681 (versus 6513 for native BPTI), the expected mass for correctly modified BPTI. Amino acid analysis also conducted upon this protein further verified that conversion of the lysine side chains had been achieved. CRK peptide was then coupled via its terminal cysteine side chain to OMIU-BPTI using the cross-linker, SPDP. A disulfide bond between the peptide cysteine sulfhydryl group and linking reagent (shown in brackets below) is formed to produce the final protein-peptide conjugate: BPTI-NH-{CO-CH2-CH2-S}-S-Cys-RK Peptide. “BPTI-CRK” is the abbreviation used to denote CRK peptide conjugated to OMIU-BPTI. The conjugate was then purified using an FPLC and a Mono-S column. The conjugate was eluted using a 0–100% 1m NaCl gradient in 100 mm sodium phosphate, pH 7.5. The conjugate eluted at ∼0.95 m NaCl with this gradient and the fraction collected at this salt concentration was then dialyzed against 0.1 m NaCl followed by de-ionized, distilled water via ultrafiltration and an Amicon YM-3 membrane. NMR samples of CRK peptide, OMIU-BPTI, and BPTI-CRK peptide conjugate were prepared in 90% H2O, 10% D2O solutions at pH 4.1 with typical sample volumes of 0.5 ml. The various sample concentrations were 5.7 mm(CRK), 6.0 mm (modified BPTI), and 5.9 mm (BPTI-CRK). The pH of all NMR samples (uncorrected for deuterium isotope effects) was adjusted to 4.1 using DCl and NaOD solutions and an Orion520A pH meter. All proton resonances were ultimately referenced against DSS. To maintain the uncoupled cysteinylated peptide in its reduced state, dithiothreitol-d 6 was added to these samples using a molar ratio of dithiothreitol:peptide = 50:1. A Bruker DMX 500MHz spectrometer was used to acquire the NMR data presented. Standard NMR experiments and pulse sequences such as DQF-COSY (35Rance M. Sørensen O.W. Bodenhausen G. Ernst R.R. Wüthrich K. Biochem. Biophys. Res. Commun. 1983; 117: 479-485Crossref PubMed Scopus (2597) Google Scholar, 36Marion D. Wüthrich K. Biochem. Biophys. Res. Commun. 1983; 113: 967-974Crossref PubMed Scopus (3524) Google Scholar), NOESY (37Jeener J. Meier B.H. Bachmann P. Ernst R.R. J. Chem. Phys. 1979; 71: 4546-4553Crossref Scopus (4838) Google Scholar), TOCSY (38Braunschweiler L. Ernst R.R. J. Magn. Reson. 1983; 53: 521-528Crossref Scopus (3108) Google Scholar), and double quantum experiments (39Rance M. Wright P.E. J. Magn. Reson. 1986; 66: 372-378Google Scholar, 40Rance M. Chazin W.J. Dalvit C. Wright P.E. Methods Enzymol. 1989; 176: 114-134Crossref PubMed Scopus (43) Google Scholar) were used for assignment purposes. The water resonance was suppressed via the use of low level rf pulses applied at the beginning of the pulse sequence (usually 1.5 s long) and during the mixing time of a NOESY sequence. The sweep widths used were 5000–8012 Hz. The number of points collected was 8192 or 4096 during t 2 and 512 or 1024 points duringt 1. For each t 1 point, 32 scans were acquired for all the NMR experiments. All NMR probe temperatures were calibrated using neat methanol (41Van Geet A.L. Anal. Chem. 1970; 42: 679-680Crossref Scopus (942) Google Scholar). The NMR data were processed using Felix 2.30 (Biosym Technologies, Inc.) software run either on a Silicon Graphics INDIGO R4000 or a Sun SPARCstation5 computer. Typical processed two-dimensional data matrices were 4 K by either 1 or 2 K. A π/2 phase-shifted sinebell window function was applied during F1 and F2 processing. Prior to F1 transformation, the first interferogram was multiplied by 0.5 to suppress t 1 ridges (42Otting G. Wider H. Wagner G. Wüthrich K. J. Magn. Reson. 1986; 66: 187-193Google Scholar). The intensities of NOESY cross-peaks were measured by calculating the volumes of the cross-peaks as the integral of all the data point intensities within the cross-peak footprints. For the NOE intensity measurement of free and coupled peptides, the relative intensities of these NOEs were calibrated against thed αN(i, i+1) NOE observed between the F10 Hα and V11 HN protons of each peptide. Antibodies 5023A and 5025A were cleaved using papain and purified following standard procedures (43Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988: 628-629Google Scholar). All buffers were degassed before use and most steps in the procedure were conducted under N2 gas. The enzyme and antibody solutions were mixed in a 1:100 papain:antibody ratio. The cleavage proceeded at 310 K for 12 h before the addition of iodoacetamide to a final concentration of 40 mm. This mixture was then incubated at 298 K for 1 h. The Fab was then dialyzed against a phosphate-buffered saline, pH 7.0, buffer using an Amicon and a YM30 membrane. The Fab fragment was purified with a FPLC system equipped with a Pharmacia 5-ml pre-packed protein A column using a pH 7.0 phosphate-buffered saline wash buffer and a 50 mm citrate, pH 3.0, elution buffer. Binding kinetics were determined by SPR using a BIAcore 1000TM biosensor system (Biacore Inc., Piscataway, NJ) (44Jonsson U. Fagerstam L. Ivarsson B. Johnsson B. Karlsson R. Lundh K. Lofas S. Persson B. Roos H. Ronnberg I. Sjolander S. Stenberg E. Stahlberg R. Urbaniczky C. Ostlin H. Malmqvist M. BioTechniques. 1991; 11: 620-627PubMed Google Scholar). The BPTI-peptide conjugate and gp120 (strain LAV, obtained from MicroGeneSys, Inc. via the NIH AIDS Research and Reference Reagent Program) were immobilized on research grade CM5 sensor chips at concentrations of 5 mg/ml in 10 mm sodium acetate, pH 4.5, for the conjugate and 10 mm sodium acetate, pH 6, for gp120 using the amine coupling kit supplied by the manufacturer. Approximately 150 resonance units of each conjugate and 2500 resonance units of gp120 were immobilized; one RU corresponds to an immobilized protein concentration of ∼1 pg/mm2 (45Stenberg E. Persson B. Roos H. Urbaniczky C. J. Coll. Interface Sci. 1991; 143: 513-526Crossref Scopus (1007) Google Scholar). Unreacted moieties on the surface were blocked with ethanolamine. The N-terminal cysteine of CRK was used to immobilize the peptide onto research grade CM5 sensor chips using a thiol-exchange coupling procedure. The carboxylated dextran surfaces of the sensor chips were first activated with N-hydroxysuccinimide andN-ethyl-N′-(dimethylaminopropyl)carbodiimide. Reactive disulfides were then introduced by reaction with 2-(2-pyridinyldithio)ethaneamine in 100 mm borate buffer, pH 8.5. The N-terminal cysteinylated peptide was then introduced at concentrations of 5 and 0.5 mg/ml, respectively, in 10 mmacetate buffer, pH 4.5. Finally, unreacted moieties on the surface were blocked with cysteine. All measurements of 5023A and 5025A Fab binding to the conjugate, gp120, and peptide surfaces were carried out in HEPES-buffered saline which contained 10 mm HEPES, pH 7.4, 150 mm NaCl, 3.4 mm EDTA. Analyses were performed at 298 K and flow rates of 30–50 ml/min. All surfaces were regenerated with 100 mm H3PO4. Association and dissociation rate constants were calculated by numerical integration and global fitting to a 1:1 interaction model using BIAevaluation 3.0 software (Biacore, Inc.) and the equation: dRU(t)/dt =k a C(R max − RU(t)) − k dRU(t), where RU(t) is the response at time t,R max is the maximum response, C is the concentration of analyte in solution, k a is the association rate constant, k d is the dissociation rate constant, and RU (0) = 0. Proton chemical shift assignments for uncoupled and BPTI-coupled CRK peptide (TableI) were made on the basis of DQF-COSY, TOCSY, and NOESY data obtained from these samples according to standard assignment procedures (46Wüthrich K. NMR of Proteins and Nucleic Acids. John Wiley & Sons, New York, NY1986Crossref Google Scholar). The spin systems were established from DQF-COSY and TOCSY data, while sequential resonance assignments were determined from NOESY data collected on these samples. All resonance assignments reported here for the various peptide and BPTI forms were obtained at 298 K and pH 4.1.Table IFree and conjugated CRK 1 H chemical shift assignmentsResidueNHαHβHOthersC-Cys−14.393.27 & 3.18Cys−14.293.12C-Arg18.944.491.79γH 1.62 δH 3.20 NɛH 7.23Arg18.814.471.82γH 1.66 δH 3.22 NɛH 7.21 *NηH 6.69C-Ile28.384.171.81γH 1.50 & 1.22 γCH3 0.94 δCH3 0.86Ile28.384.221.84γH 1.51 & 1.22 γCH3 0.90 δCH3 0.87C-Gln38.574.402.05 & 1.96γH 2.33 NɛH 7.52 & 6.88Gln38.574.422.09 & 1.99γH 2.36 NɛH 7.54 & 6.88C-Arg48.514.411.82γH 1.63 δH 3.18 NɛH 7.19Arg48.494.431.88 & 1.78γH 1.67 δH 3.21 NɛH 7.21 *NηH 6.69C-Gly58.354.17 & 4.068.314.05 & 3.84Gly58.344.30 & 4.058.314.09 & 3.85C-Pro64.462.27γH 2.03 δH 3.644.632.39 & 2.17γH1.87 δH 3.60 & 3.53Pro64.482.30 & 2.00γH 2.06 & 2.01 δH 3.674.642.40γH1.97 & 1.89 δH 3.57C-Gly78.523.958.644.00Gly78.523.978.644.02C-Arg88.164.301.82 & 1.72γH 1.59 δH 3.17 NɛH 7.198.344.31Arg88.164.331.82 & 1.74γH 1.61 & 1.63 δH 3.20 & 3.218.344.331.85 & 1.72NɛH 7.18 *NηH 6.69C-Ala98.284.291.31Ala98.274.311.32C-Phe108.204.663.11 & 3.032,6H 7.22 3,5H 7.34 4H 7.28Phe108.194.673.15 & 3.032,6H 7.25 3,5H 7.37 4H 7.30C-Val118.084.192.03γH 0.90Val118.084.212.05γH 0.93C-Thr128.294.384.14γH 1.20Thr128.284.354.16γH 1.21C-Ile138.264.221.80γH 1.50 & 1.22 γCH3 0.87 δCH3 0.86Ile138.254.231.90γH 1.52 & 1.24 γCH3 0.96 δCH3 0.89C-Gly148.503.97Gly148.493.98C-Lys157.894.231.84γH 1.39 δH 1.68ɛH 3.00 NH3+ 7.53Lys157.874.231.86γH 1.41 δH 1.71 ɛH 3.02 NH3+ 7.54 εH 3.02 NH3+7.54The proton resonances (in ppm) of free and conjugated CRK were assigned at pH 4.1 and 298 K. The free and conjugated CRK residues are represented using standard three-letter amino acid abbreviations, followed by N, where N corresponds to the residue number. Free and conjugated CRK residues are represented as “XxxN” and “CXxxN”, respectively. The H2O resonance (4.8 ppm at 298 K) is used as the internal frequency standard. Degenerate protons are indicated using a single chemical shift value. The chemical shift values of cis form resonances are italicized. Asterisk indicates that all the arginine NηH protons of free CRK were unresolvable. Overlap with BPTI resonances prevented assignment of the following conjugated CRK resonances: C-Arg8 (cis form) side chain and all arginine NηH protons. Open table in a new tab The proton resonances (in ppm) of free and conjugated CRK were assigned at pH 4.1 and 298 K. The free and conjugated CRK residues are represented using standard three-letter amino acid abbreviations, followed by N, where N corresponds to the residue number. Free and conjugated CRK residues are represented as “XxxN” and “CXxxN”, respectively. The H2O resonance (4.8 ppm at 298 K) is used as the internal frequency standard. Degenerate protons are indicated using a single chemical shift value. The chemical shift values of cis form resonances are italicized. Asterisk indicates that all the arginine NηH protons of free CRK were unresolvable. Overlap with BPTI resonances prevented assignment of the following conjugated CRK resonances: C-Arg8 (cis form) side chain and all arginine NηH protons. In the uncoupled and coupled CRK peptide spectra (Table I), a major, as well as minor, set of resonances was observed for residues Gly5 to Arg8. Based upon thed ααα(i,i+1) NOEs observed between the Hα protons of the minor form of Gly5 and Pro6 that were measured from D2O samples of free CRK and peptide (data not shown), the minor resonances were attributed to the cis form of these peptides arising from cis-trans isomerization about the Gly5-Pro6 peptide bond. The amount ofcis conformer present in both peptide forms was estimated to be close to 10%. Nearly complete proton resonance assignments for native, 2Upon request, we will provide the 1H NMR assignments for native, OMIU-modified, and peptide-conjugated BPTI. Peptide J-coupling, temperature coefficient, and ROESY data will also be given upon request. OMIU-modified, and peptide-conjugated BPTI were obtained in order to determine whether chemical modification and peptide coupling significantly affected BPTI. This was accomplished via comparison of the native BPTIversus OMIU-BPTI HN and Hα resonance frequencies. Very few resonance frequency changes were observed for most BPTI residues regardless of BPTI form and most of these were less than 0.02 ppm. The largest changes (observed after linkage of SPDP to OMIU-modified BPTI) consisted of large frequency shifts involving the following protein resonances: the HN and Hα of Ala58 and the Hα of R1. The OMIU-modified Ala58 backbone resonances underwent substantial frequency changes due to peptide coupling: the NH, Hα resonances shifted from 8.05 (4.05) to 7.81 (4.13 ppm) after coupling. A corresponding R1 Hα frequency shift from 4.36 ppm (OMIU-BPTI) to 4.58 ppm (BPTI-CRK) was also seen. Comparison of the modified versus peptide-conjugated BPTI R1 amide HN frequency was not possible since the R1 amino group is converted to an amide after peptide coupling. The uncoupled peptide gave rise to medium-strong dαN(i, i+1) NOEs involving residues Arg1 through Gly14 (Fig.1A). A weakd αN(i, i+1) NOE was also observed between Arg1 and Ile2. At the same time, a few medium to weakd NN(i, i+1) NOEs were detected between residues Gly7-Thr12. Other sequential NOEs observed included ad βN(i, i+1) NOE between Ala9 and Phe10. In addition, strongd αδ(i,i+1) NOEs between both Gly5 Hα protons and the Pro6 Hδ proton were measured. Intraresidued Nα(i, i) NOEs were observed for residues Gly5, Gly7, Val11, Thr12, and Gly14. This type of NOE was also observed for Arg8. The intraresidued Nα(i, i) NOEs of Arg4 and Ala9 were overlapped with sequentiald αN(i, i+1) NOEs between Gln3-Arg4 and Arg8-Ala9, respectively. As observed for the uncoupled peptide, most of the backbone protons of the coupled peptides gave rise to strongd αN(i, i+1) NOEs (Fig. 1 B). For coupled CRK peptide,d NN(i, i+1) NOEs were measured between sequential residues beginning with Ile2-Thr12 (except Gln3-Arg4 and Ala9-Phe10). The strongestd NN(i, i+1) NOEs involved residues Gly7-Ala9. Furthermore, a weak Pro6-Arg8dαN(i, i+2) NOE was observed (Fig. 2 A). For residue Pro6, strongd αδ(i,i+1) NOEs between Gly5 and Pro6 were observed. Additionally, a d Nδ(i,i+1) NOE between the Gly5 HN and Pro6 Hδ protons was observed for coupled CRK. Another weak, but significantd δN(i, i+1) NOE between the Pro6 Hδ and Gly7 HN protons was also detected (Fig. 2 B). Other sequential NOEs observed included d βN(i,i+1) NOEs between residues Ile2-Gln3, Ala9-Phe10, and Val11-Thr12. For the conjugated peptide, strong to medium intraresidued Nα(i+1) NOEs were measured, except for residue Phe10, for which a weak NOE of this type was measured instead. This type of NOE was not observed for Cys−1 and Arg1. Intraresidued Nα (i+1) NOEs of Arg4 and Ala9 were overlapped withd αN(i, i+1) NOEs of Gln3-Arg4 and Arg8-Ala9, respectively. The corresponding Ile13 NOE was overlapped with an intramolecular BPTI NOE. The β−turn NOEs observed for conjugated CRK were absent in the corresponding free peptide NOESY data, regardless of the mixing time (100–600 ms) used. To verify whether this discrepancy was a consequence of the peptide correlation time, free peptide ROESY data2 were also collected (data not shown). The overall pattern of peptide resonanc" @default.
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• W2006756069 title "The Binding of a Glycoprotein 120 V3 Loop Peptide to HIV-1 Neutralizing Antibodies" @default.
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