FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2065572647> ?p ?o ?g. }

• W2065572647 endingPage "31411" @default.
• W2065572647 startingPage "31407" @default.
• W2065572647 abstract "The Fv fragment of the 0.5β monoclonal antibody has recently been constructed, expressed, and purified. It binds with nanomolar affinity to the immunogenic RP135 peptide that is derived from the principal neutralizing determinant of HIV-1 in the third hypervariable region of gp120. Here, we analyzed the temperature-dependence of binding of the 0.5β Fv fragment to the RP135 peptide and a series of mutants thereof. Our results show that there is almost complete enthalpy-entropy compensation in the effects of mutations in the peptide on binding to the Fv, indicating that the mutations do not change the binding mechanism. There is good correlation, for residues within the antigenic epitope, between mutational effects on ΔC p and calculated values of ΔΔC p based on the extent of burial of polar and non-polar surface areas of amino acids. The value of ΔC p for the binding of the 0.5β Fv fragment to the wild-type RP135 peptide is found to be −5.0 (± 0.9) kcal K−1 mol−1 in the presence of 0.1% Tween-20 but only −0.1 (± 0.9) kcal K−1mol−1 in its absence. This result has important implications for the successful application of the structural parameterization approach to predicting changes in heat capacity that accompany binding reactions carried out in the presence of detergent or protein-stabilizing agents. The Fv fragment of the 0.5β monoclonal antibody has recently been constructed, expressed, and purified. It binds with nanomolar affinity to the immunogenic RP135 peptide that is derived from the principal neutralizing determinant of HIV-1 in the third hypervariable region of gp120. Here, we analyzed the temperature-dependence of binding of the 0.5β Fv fragment to the RP135 peptide and a series of mutants thereof. Our results show that there is almost complete enthalpy-entropy compensation in the effects of mutations in the peptide on binding to the Fv, indicating that the mutations do not change the binding mechanism. There is good correlation, for residues within the antigenic epitope, between mutational effects on ΔC p and calculated values of ΔΔC p based on the extent of burial of polar and non-polar surface areas of amino acids. The value of ΔC p for the binding of the 0.5β Fv fragment to the wild-type RP135 peptide is found to be −5.0 (± 0.9) kcal K−1 mol−1 in the presence of 0.1% Tween-20 but only −0.1 (± 0.9) kcal K−1mol−1 in its absence. This result has important implications for the successful application of the structural parameterization approach to predicting changes in heat capacity that accompany binding reactions carried out in the presence of detergent or protein-stabilizing agents. Advances in antibody technology have made possible direct cloning of antibody genes from hybridomas or lymphocytes into plasmid vectors, thereby facilitating protein engineering studies upon their expression in bacteria (1Winter G. Milstein C. Nature. 1991; 349: 293-299Crossref PubMed Scopus (846) Google Scholar, 2Plückthun A. Immunol. Rev. 1992; 130: 151-188Crossref PubMed Scopus (129) Google Scholar, 3Nilsson B. Curr. Opin. Struct. Biol. 1995; 5: 450-456Crossref PubMed Scopus (16) Google Scholar). Using protein engineering methods, one may tailor the specificity of antibody fragments, enhance their binding activity, and humanize them for use in therapy (4Rees A.R. Staunton D. Webster D.M. Searle S.J. Henry A.H. Pedersen J.T. Trends Biotechnol. 1994; 12: 199-206Abstract Full Text PDF PubMed Scopus (41) Google Scholar, 5Hurle M.R. Gross M. Curr. Opin. Biotechnol. 1994; 5: 428-433Crossref PubMed Scopus (24) Google Scholar). Recently (6Faiman G.A. Levy R. Anglister J. Horovitz A. J. Biol. Chem. 1996; 271: 13829-13833Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar), we constructed, expressed, and purified the Fv fragment of the 0.5β monoclonal antibody. This antibody, which was raised against the envelope glycoprotein gp120 from the IIIB isolate of human immunodeficiency virus type 1 (HIV-1) 1The abbreviations used are: HIV, human immunodeficiency virus; NMR, nuclear magnetic resonance. (7Matsushita S. Robert-Guroff M. Rusche J. Koito A. Hattori T. Hoshino H. Javaherian K. Takatsuki K. Putney S. J. Virol. 1988; 62: 2107-2114Crossref PubMed Google Scholar), binds to the principal neutralizing determinant of HIV-1. The 0.5β Fv fragment binds with nanomolar affinity (6Faiman G.A. Levy R. Anglister J. Horovitz A. J. Biol. Chem. 1996; 271: 13829-13833Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar) to the immunogenic 24-amino acid long RP135 peptide, NNTRKSIRIQRGPGRAFVTIGKIG (8Palker 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 (442) Google Scholar), which is derived from the principal neutralizing determinant of HIV-1 found in the third hypervariable region of gp120. Recent nuclear magnetic resonance (NMR) investigations defined a 16-amino acid long antigenic epitope from Lys5 to Ile20 in the RP135 peptide (corresponding to residues 312–327 in gp120) and showed that it forms an 8-residue turn upon binding to the 0.5β monoclonal antibody (9Zvi A. Kustanovich I. Feigelson D. Levy R. Eisenstein M. Matsushita S. Richalet-Sécordel P. Regenmortel M.H.V. Anglister J. Eur. J. Biochem. 1995; 229: 178-187Crossref PubMed Scopus (44) Google Scholar,10Zvi A. Feigelson D.J. Hayek Y. Anglister J. Biochemistry. 1997; 36: 8619-8627Crossref PubMed Scopus (34) Google Scholar). The crystal and solution structures of the 0.5β monoclonal antibody (or fragments thereof) without and in complex with the peptide antigen have not yet been solved. The structures of antibody fragments of two other monoclonal antibodies, 50.1 and 59.1, in complex with V3 loop-derived peptides have, however, been determined (11Rini J.M. Stanfield R.L. Stura E.A. Salinas P.A. Profy A.T. Wilson I.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6325-6329Crossref PubMed Scopus (213) Google Scholar, 12Ghiara J.B. Stura E.A. Stanfield R.L. Profy A.T. Wilson I.A. Science. 1994; 264: 82-85Crossref PubMed Scopus (240) Google Scholar). In both cases, the antibody fragments bind 7-residue-long epitopes in a β-turn conformation, as frequently found in other antibody-peptide interactions (13Stanfield R.L. Wilson I.A. Curr. Opin. Struct. Biol. 1995; 5: 103-113Crossref PubMed Scopus (150) Google Scholar). Analysis of the energetics of peptide-antibody interactions is complicated by the fact that peptide antigens often undergo large conformational changes upon binding. To date, the energetics of interaction between antibodies and HIV-1-derived peptide antigens have been little studied. Previously (6Faiman G.A. Levy R. Anglister J. Horovitz A. J. Biol. Chem. 1996; 271: 13829-13833Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar), we characterized the salt dependence of the interaction between the 0.5β Fv fragment and the RP135 peptide and measured the contribution of arginine residues in the peptide to the interaction. To obtain further structural information on the interaction between the 0.5β Fv fragment and the RP135 peptide, we decided to determine the temperature dependence of the binding constant of this reaction for wild-type peptide and six mutants thereof. Isothermal calorimetry experiments did not yield reproducible results, probably owing to aggregation and/or nonspecific binding. We therefore carried out a van't Hoff analysis in the presence of the non-ionic detergent Tween-20. Our results show that there is enthalpy-entropy compensation in the effects of the mutations in the peptide on the binding to the Fv fragment, indicating that the mutations do not change the binding mechanism. Using van't Hoff plots, one can estimate the change in heat capacity, ΔC p, associated with the binding reaction. We find that the value of ΔC p depends strongly on the concentration of detergent. This finding has important implications for attempts to correlate values of ΔC p with the amount of surface area buried in protein-ligand complexes (14Murphy K.P. Freire E. Adv. Protein Chem. 1992; 43: 313-361Crossref PubMed Google Scholar, 15Spolar R.S. Record Jr., M.T. Science. 1994; 263: 777-784Crossref PubMed Scopus (1373) Google Scholar). Although such a correlation has been applied successfully to predict the amount of surface area buried in the interface of some protein-protein complexes (16Murphy K.P. Freire E. Paterson Y. Proteins Struct. Funct. Genet. 1995; 21: 83-90Crossref PubMed Scopus (79) Google Scholar, 17Murphy K.P. Xie D. Garcia K.C. Amzel L.M. Freire E. Proteins Struct. Funct. Genet. 1993; 15: 113-120Crossref PubMed Scopus (191) Google Scholar, 18Hibbits K.A. Gill D.S. Willson R.C. Biochemistry. 1994; 33: 3584-3590Crossref PubMed Scopus (82) Google Scholar), it has failed in the case of others (19Mandiyan V. O'Brien R. Zhou M. Margolis B. Lemmon M.A. Sturtevant J.M. Schlessinger J. J. Biol. Chem. 1996; 271: 4770-4775Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 20Varadarajan R. Connelly P.R. Sturtevant J.M. Richards F.M. Biochemistry. 1992; 31: 1421-1426Crossref PubMed Scopus (104) Google Scholar, 21De Cristofaro R. Landolfi R. J. Mol. Biol. 1994; 239: 569-577Crossref PubMed Scopus (21) Google Scholar, 22Ayala Y.M. Vindigni A. Nayal M. Spolar R.S. Record Jr., M.T. Di Cera E. J. Mol. Biol. 1995; 253: 787-798Crossref PubMed Scopus (47) Google Scholar, 23Guinto E.R. Di Cera E. Biochemistry. 1996; 35: 8800-8804Crossref PubMed Scopus (86) Google Scholar, 24Ladbury J.E. Wright J.G. Sturtevant J.M. Sigler P.B. J. Mol. Biol. 1994; 238: 669-681Crossref PubMed Scopus (193) Google Scholar). We suggest that this failure is due, in part, to the frequent use of protein-stabilizing agents or detergents in binding experiments. Expression and purification of the 0.5β Fv fragment was achieved as before (6Faiman G.A. Levy R. Anglister J. Horovitz A. J. Biol. Chem. 1996; 271: 13829-13833Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). The antigenic peptide TRKSIRIQRGPGRAFVTIGK and variants thereof were synthesized, purified, and characterized as described (6Faiman G.A. Levy R. Anglister J. Horovitz A. J. Biol. Chem. 1996; 271: 13829-13833Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). The concentrations of the peptides were estimated using the 2,4,6-trinitrobenzenesulfonic acid method for determination of amines (25Snyder S.L. Sobocinski P.Z. Anal. Biochem. 1975; 64: 284-288Crossref PubMed Scopus (756) Google Scholar). Isopropyl β-d-thiogalactopyranoside was obtained from Chembridge Corp., Illinois. All other analytical grade reagents were purchased from Sigma. Fluorescence measurements of the 0.5β Fv fragment in the presence of different peptide concentrations were made as described previously (6Faiman G.A. Levy R. Anglister J. Horovitz A. J. Biol. Chem. 1996; 271: 13829-13833Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). All the reactions were carried out in 50 mm sodium phosphate buffer (pH 7.5) containing different fixed amounts of the non-ionic detergent Tween-20. Phosphate buffers were prepared separately for each temperature using a pK a value of 6.86 at 25 °C and a temperature-dependence coefficient, ∂pK a /∂T, of −0.0028 (26Stoll V.S. Blanchard J.S. Methods Enzymol. 1990; 182: 24-38Crossref PubMed Scopus (196) Google Scholar). The titrations were carried out at least in duplicate over a temperature range from 10 to 35 °C (± 0.1 °C) in a thermostatted cuvette. Differential scanning calorimetry was carried out using the MCS instrument (Microcal Inc., Amherst, MA) at a scan rate of 1 K min−1. The concentration of the Fv fragment was 26 μm in 50 mm phosphate buffer (pH 7.5) without or with 0.05% Tween-20. Determination of dissociation constants (K d ) between the Fv fragment and the different peptides from the fluorescence enhancement titrations was achieved as described previously (6Faiman G.A. Levy R. Anglister J. Horovitz A. J. Biol. Chem. 1996; 271: 13829-13833Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). van't Hoff analysis was carried out by plotting ln K a (= ln(1/K d )) as a function of the inverse absolute temperature (1/T). The data were fitted using Kaleidagraph (Version 2.1, Synergy Software (PCS Inc.)) to the following equation,lnKa=−ΔH0R1T−1T0+ΔCpT0R1T−1T0+ΔCpRlnTT0+lnKa,0Equation 1 where K a is the association constant,T 0 is a reference temperature in degrees Kelvin,K a ,0 and ΔH 0are the association constant and enthalpy change at the reference temperature, and ΔC p is the heat capacity change of the binding reaction. Estimates (± S.E.) of the parameters ΔH 0, ΔC p, and ln K a ,0 were obtained from the fits. The changes in free energy of binding, ΔG 0, and entropy, ΔS 0, at the reference temperature of 298 K were calculated using the following relations,ΔG0=ΔH0−TΔS0=−RT lnKa,0Equation 2 where R is the gas constant, and all other notations are as before. Equation 1 is derived by combining Equation 2 and the following relations.ΔCp=∂ΔH/∂T=T(∂ΔS/∂T)Equation 3 Solvent-accessible areas were calculated using the method of Lee and Richards (27Lee B. Richards F.M. J. Mol. Biol. 1971; 55: 379-400Crossref PubMed Scopus (5360) Google Scholar) in the Homology package within Insight II (Biosym/MSI). A linear dependence of the fluorescence on the Fv concentration at different temperatures (in the absence of peptide) indicates that dissociation of the Fv fragment into light and heavy chains is negligible (Fig. 1). The thermodynamic parameters of the interaction between the 0.5β Fv fragment and the RP135 peptide at different temperatures between 10 and 35 °C were determined in the presence of different concentrations of Tween-20 by monitoring fluorescence enhancement upon binding. Plots of the natural logarithm of the association constant as a function of the inverse absolute temperature were found to have increasingly more convex shape in the presence of increasing concentrations of the non-ionic detergent (Fig. 2). Convex plots are characteristic of processes with a negative change in heat capacity. The data were fitted using Equation 1 and 298 K as a reference temperature. A linear relationship is observed between the value of ΔC p and the concentration of Tween-20 with a correlation coefficient of 0.89 (Fig.3). The value of ΔC p in the absence of detergent is estimated by linear extrapolation to be −0.1 (± 0.9) kcal K−1 mol−1.Figure 2van't Hoff plots for the binding of wild-type and mutant RP135 peptides to the 0.5β Fv fragment.Binding experiments were carried out at different temperatures in 50 mm sodium phosphate buffer (pH 7.5) containing different concentrations of Tween-20 in the case of wild-type peptide (panel A) and 0.05% Tween-20 in the case of the mutants (panels B-D). For additional details, see “Experimental Procedures.” The data were fitted to Equation 1.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Plot of the change in heat capacity upon binding of wild-type RP135 peptide to the 0.5β Fv fragment as a function of the concentration of Tween-20. Binding experiments were carried out in the presence of different fixed concentrations of Tween-20 as described under “Experimental Procedures.” A linear relationship is observed with a correlation coefficient of 0.89.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Differential scanning calorimetry of the 0.5β Fv fragment was carried out in the absence or in the presence of 0.05% Tween-20 (Fig. 4). Of importance is that the apparent heat capacity of the Fv fragment is always less negative when Tween-20 is present. In the absence of Tween-20, two transitions are observed that may correspond to the dissociation of the Fv fragment and the denaturation of the two chains. In the presence of Tween-20, a third transition is observed that may be due to different melting temperatures of the light and heavy chains when the detergent is present. Six variants of the RP135 peptide with the single amino acid replacements R4A, K5A, R8A, R11A, F17A, and T19A were used in this study. These mutations span the entire sequence of the RP135 peptide except for the conserved GPGR loop that is thought to be crucial for stabilizing the conformation of the peptide (12Ghiara J.B. Stura E.A. Stanfield R.L. Profy A.T. Wilson I.A. Science. 1994; 264: 82-85Crossref PubMed Scopus (240) Google Scholar, 28Zvi A. Hiller R. Anglister J. Biochemistry. 1992; 31: 6972-6979Crossref PubMed Scopus (67) Google Scholar). van't Hoff plots were constructed for the binding of these RP135 variants to the 0.5β Fv fragment in the presence of 0.05% Tween-20 (Fig. 2). The changes in heat capacity upon binding of the mutant peptides to the Fv were found to be negative and relatively large in all of the cases (TableI). The effects of the replacements R4A, K5A, R8A, R11A, F17A, and T19A on the change in heat capacity upon binding (ΔC p(wild-type) − ΔC p(mutant)) were found to be −0.7 (± 1.1), −0.2 (± 1.4), −2.6 (± 0.9), −2.3 (± 1.1), −1.8 (± 1.0), and −2.0 (± 0.9) kcal K−1 mol−1, respectively. The free energies, enthalpies, and entropies of binding of wild-type and mutant peptides at the reference temperature of 298 K were calculated using Equation 2 and are given in Table I. The effects of the replacements R4A, K5A, R8A, R11A, F17A, and T19A on the enthalpies of binding at 298 K (ΔH 0(wild-type) − ΔH 0(mutant)) are calculated to be 2.4 (± 3.2), −2.3 (± 5.2), −8.9 (± 1.1), −2.4 (± 3.5), −0.5 (± 2.3), and −9.1 (± 1.3) kcal mol−1, respectively. The effects of the replacements R4A, K5A, R8A, R11A, F17A, and T19A on the entropies of binding at 298 K (ΔS 0(wild-type) − ΔS 0(mutant)) are calculated to be 7 (± 11), −8 (± 18), −16 (± 4), −3 (± 12), 5 (± 9), and −27 (± 4) cal K−1 mol−1, respectively. The effects of the replacements R4A, K5A, R8A, R11A, F17A, and T19A on the free energies of binding at 298 K (ΔG 0(wild-type) − ΔG 0(mutant)) are calculated to be 0.16 (± 0.11), 0.09 (± 0.18), −4.14 (± 0.04), −1.45 (± 0.12), −1.86 (± 0.09), and −0.90 (± 0.04) kcal mol−1, respectively.Table IThermodynamic parameters of the interaction at 25 °C between the 0.5β Fv fragment and the RP135 peptide or mutants thereofPeptideΔG0ΔH 0ΔS 0TΔS 0ΔC pkcal mol −1kcal mol −1cal K −1 mol −1kcal mol −1kcal K −1 mol −1Wild-type−11.46 ± 0.03−22.1 ± 0.9−36 ± 3−10.7 ± 0.9−3.4 ± 0.9R4A−11.63 ± 0.10−24.5 ± 3.1−43 ± 10−12.9 ± 3.1−2.7 ± 0.7K5A−11.55 ± 0.17−19.9 ± 5.2−28 ± 17−8.3 ± 5.2−3.2 ± 1.1R8A−7.33 ± 0.02−13.3 ± 0.5−20 ± 2−6.0 ± 0.5−0.8 ± 0.1R11A−10.01 ± 0.11−19.8 ± 3.4−33 ± 11−9.7 ± 3.4−1.1 ± 0.7F17A−9.61 ± 0.08−21.7 ± 2.4−40 ± 8−12.0 ± 2.4−1.6 ± 0.5T19A−10.56 ± 0.03−13.1 ± 0.9−8 ± 3−2.5 ± 0.9−1.4 ± 0.2All the titrations were carried out at least in duplicate in 50 mm sodium phosphate buffer (pH 7.5) containing 0.05% Tween-20 as described under “Experimental Procedures.” Open table in a new tab All the titrations were carried out at least in duplicate in 50 mm sodium phosphate buffer (pH 7.5) containing 0.05% Tween-20 as described under “Experimental Procedures.” The entropies of binding of wild-type and mutant peptides to the Fv fragment at 298 K and 0.05% Tween-20 were plotted as a function of the respective enthalpies of binding (Fig. 5). The data were fitted to the linear equation,ΔSi=α+βΔHiEquation 4 where i stands for the ith peptide. A linear relationship is observed with a slope, β, of 0.0026 (± 0.0004) K−1 and a correlation coefficient of 0.94. This result indicates almost complete entropy-enthalpy compensation. The change in solvent-accessible area upon binding of the RP135 peptide to the 0.5β Fv fragment is due to (i) area buried within the peptide and (ii) area buried in the interface between the peptide and Fv in their complex. The area buried within the peptide is the difference between the solvent-accessible area of the free peptide in its bound and unbound (extended) conformations. The conformation of a shorter version (Arg4-Ile20) of the peptide in its bound state was recently determined by NMR methods (10Zvi A. Feigelson D.J. Hayek Y. Anglister J. Biochemistry. 1997; 36: 8619-8627Crossref PubMed Scopus (34) Google Scholar). The solvent-accessible area of the free peptide in its bound conformation was calculated using the NMR-derived coordinates to be 2064.8 Å2 (of which 1048.7 Å2 are nonpolar and 1016.1 Å2 are polar). The solvent-accessible area of the shorter version of the free peptide in an extended conformation was calculated to be 2444.7 Å2 (of which 1364.7 Å2 are nonpolar and 1080.0 Å2 are polar). The area buried within the peptide upon binding to the Fv fragment is, therefore, 379.9 Å2(of which 316.0 Å2 are nonpolar and 63.9 Å2are polar). In the absence of a known structure of the complex, the interface area between the peptide and the Fv in the complex was estimated from the literature on other peptide-antibody complexes (13Stanfield R.L. Wilson I.A. Curr. Opin. Struct. Biol. 1995; 5: 103-113Crossref PubMed Scopus (150) Google Scholar) to be about 1000 Å2 (of which about 500 Å2are estimated to be nonpolar and 500 Å2 are polar). The interaction between the RP135 peptide and the 0.5β Fv fragment was studied here by measuring the effects of mutations in the peptide on the temperature-dependence of the binding reaction. In principle, the mutations in the peptide may alter the binding mechanism, thereby considerably complicating the analysis of their effects. It can be shown that enthalpy-entropy compensation is a good indication for a single interaction mechanism (29Leffler J.E. Grunwald S. Rates and Equilibria of Organic Reactions. J. Wiley & Sons, New York1963: 315-342Google Scholar). The almost complete enthalpy-entropy compensation observed here (Fig. 5) indicates, therefore, a single interaction mechanism of the different mutant peptides with the Fv fragment. Heat capacity changes associated with peptide binding are usually in the range of −0.1 to −0.5 kcal mol−1 K−1although some exceptions have been reported. There are several reasons for changes in heat capacity due to ligand binding. A positive contribution to the change in heat capacity is due to the decrease in configurational freedom of one or both of the molecules that form the complex. It can be readily shown that the change in heat capacity is proportional to the change in the extent of fluctuations in the system (ΔC p = {(〈H 2〉-〈H〉2)free− (〈H 2〉 − 〈H〉2)bound}/kT 2). This factor is of relatively more importance in the case of flexible peptide binding owing to the loss of side chain and backbone entropy that accompanies the binding. A negative and relatively large contribution to the change in heat capacity arises from binding-induced desolvation of nonpolar groups that are exposed to water in the unbound state. Finally, the heat capacity might change because of changes in ionization of charged side chains although this factor generally has a relatively small contribution. We have attempted to rationalize the change in heat capacity associated with the binding of the RP135 peptide to the 0.5β Fv fragment by using the previously described structure-based parameterization approach (30Gómez J. Hilser V.J. Xie D. Freire E. Proteins Struct. Funct. Genet. 1995; 22: 404-412Crossref PubMed Scopus (411) Google Scholar). According to this approach, the effect of changes in hydration and noncovalent interactions on the change in the heat capacity, ΔC p,hyd, upon folding or binding (14Murphy K.P. Freire E. Adv. Protein Chem. 1992; 43: 313-361Crossref PubMed Google Scholar, 15Spolar R.S. Record Jr., M.T. Science. 1994; 263: 777-784Crossref PubMed Scopus (1373) Google Scholar, 17Murphy K.P. Xie D. Garcia K.C. Amzel L.M. Freire E. Proteins Struct. Funct. Genet. 1993; 15: 113-120Crossref PubMed Scopus (191) Google Scholar, 31Murphy K.P. Bhakuni V. Xie D. Freire E. J. Mol. Biol. 1992; 227: 293-306Crossref PubMed Scopus (188) Google Scholar) can be expressed as a linear combination of contributions associated with changes in polar (ΔASA p) and nonpolar (ΔASA np) solvent-accessible surface areas, as follows,ΔCp,hyd=a(T)ΔASAnp+b(T)ΔASApEquation 5 where the coefficients a(T) andb(T) are the respective contributions per Å2 of nonpolar and polar areas that become solvent-exposed, which are equal to 0.45 + 2.63 × 10−4(T − 25) − 4.2 × 10−5 (T − 25)2 and −0.26 + 2.85 × 10−4(T − 25) + 4.31 × 10−5(T − 25)2 in cal K−1 mol−1 (32Gómez J. Freire E. J. Mol. Biol. 1995; 252: 337-350Crossref PubMed Scopus (226) Google Scholar). The change in solvent-accessible area upon binding of the RP135 peptide to the 0.5β Fv fragment consists of changes due to (i) area buried within the peptide and (ii) area buried in the interface of the peptide-Fv complex. The area buried within the peptide upon binding is 379.9 Å2 (of which 316 Å2 are nonpolar and 63.9 Å2 are polar). The contribution of the area buried within the peptide to ΔC p,hyd is calculated using Equation 5 to be −0.125 kcal K−1 mol−1 at 25 °C. In the absence of a known three-dimensional structure of the Fv-peptide complex, we estimated the size of the interface area by assuming that, in the complex, about half of the solvent-accessible area of the peptide becomes buried (13Stanfield R.L. Wilson I.A. Curr. Opin. Struct. Biol. 1995; 5: 103-113Crossref PubMed Scopus (150) Google Scholar). The area buried in the interface of the peptide-Fv complex is accordingly estimated to be about 1000 Å2 (of which about 500 Å2 are estimated to be nonpolar and 500 Å2 are polar) per molecule, and its contribution to ΔC p,hyd is, therefore, about −0.2 kcal K−1 mol−1. The overall contribution to ΔC p,hyd due to the change in solvent-accessible area buried within the peptide and in the interface of the peptide-Fv complex is equal to about −0.3 kcal K−1 mol−1. This value is in agreement, within experimental error, with the value of ΔC p in the absence of detergent estimated by linear extrapolation to be −0.1 (± 0.9) kcal K−1 mol−1 (Fig. 3). The magnitude of mutational effects on ΔG and ΔC p is consistent with NMR investigations (9Zvi A. Kustanovich I. Feigelson D. Levy R. Eisenstein M. Matsushita S. Richalet-Sécordel P. Regenmortel M.H.V. Anglister J. Eur. J. Biochem. 1995; 229: 178-187Crossref PubMed Scopus (44) Google Scholar,10Zvi A. Feigelson D.J. Hayek Y. Anglister J. Biochemistry. 1997; 36: 8619-8627Crossref PubMed Scopus (34) Google Scholar) that mapped the antigenic epitope from Lys5 to Ile20, i.e. replacement of Arg4 and Lys5 by alanine is found to have relatively little effect on both ΔG and ΔC p, whereas replacement by alanine of residues within the epitope has a large effect on both ΔG and ΔC p (TableI). There is a good correlation between the mutational effects on ΔC p and calculated values of ΔΔC p,hyd, using Equation 5 and the polar and nonpolar surface areas of amino acids (27Lee B. Richards F.M. J. Mol. Biol. 1971; 55: 379-400Crossref PubMed Scopus (5360) Google Scholar), for residues within the antigenic epitope (Fig. 6). Due to the uncertainty associated with linear extrapolation to zero concentration of detergent, the relatively large errors in the experimental values of ΔΔC p and the likely possibility that some side chains within the epitope are not fully buried, an agreement between the values of the experimentally measured ΔΔC p and calculated changes in ΔC p,hyd upon mutation is not to be expected. Discrepancies between measured and calculated values of ΔC p have been observed in the case of other systems (19Mandiyan V. O'Brien R. Zhou M. Margolis B. Lemmon M.A. Sturtevant J.M. Schlessinger J. J. Biol. Chem. 1996; 271: 4770-4775Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 20Varadarajan R. Connelly P.R. Sturtevant J.M. Richards F.M. Biochemistry. 1992; 31: 1421-1426Crossref PubMed Scopus (104) Google Scholar, 33Frisch C. Schreiber G. Johnson C.M. Fersht A.R. J. Mol. Biol. 1997; 267: 696-706Crossref PubMed Scopus (121) Google Scholar), suggesting that the structure-based parameterization approach may apply to folding but not to binding processes. Our results suggest that the lack of correlation is due, at least in some cases, to the presence of detergents or protein stabilizing agents. Polyethylene glycol, sucrose, and Tween-20, for example, are often used in biological assays to prevent denaturation, aggregation, or nonspecific binding. Many of these protein-stabilizing agents are excluded from the surface of proteins which, therefore, become preferentially hydrated when these compounds are present at high concentrations (34Timasheff S.N. Annu. Rev. Biophys. Biomol. Struct. 1993; 22: 67-97Crossref PubMed Scopus (979) Google Scholar). Such preferential hydration is expected to augment effects on heat capacity due to desolvation, as observed here in the case of the denaturation of the 0.5β Fv fragment in the presence of 0.05% Tween-20 (Fig. 4). Likewise, in the presence of Tween-20, both the peptide and the Fv fragment are preferentially hydrated, and complex formation between them, therefore, involves a larger desolvation step that should be reflected by a relatively more negative change in heat capacity, as indeed observed in this study (Fig. 3). This may explain why unusually large negative heat capacity changes have been observed in several other binding studies in which detergents or protein-stabilizing agents were employed (21De Cristofaro R. Landolfi R. J. Mol. Biol. 1994; 239: 569-577Crossref PubMed Scopus (21) Google Scholar, 22Ayala Y.M. Vindigni A. Nayal M. Spolar R.S. Record Jr., M.T. Di Cera E. J. Mol. Biol. 1995; 253: 787-798Crossref PubMed Scopus (47) Google Scholar, 23Guinto E.R. Di Cera E. Biochemistry. 1996; 35: 8800-8804Crossref PubMed Scopus (86) Google Scholar). We thank Dr. S. Matsushita for the 54′CB1 hybridoma cell line producing the 0.5β monoclonal antibody. We also thank R. Levy and Prof. J. Anglister for assistance and discussion during various stages of this project." @default.
• W2065572647 created "2016-06-24" @default.
• W2065572647 creator A5014486140 @default.
• W2065572647 creator A5035433072 @default.
• W2065572647 date "1997-12-01" @default.
• W2065572647 modified "2023-09-27" @default.
• W2065572647 title "Thermodynamic Analysis of the Interaction between the 0.5β Fv Fragment and the RP135 Peptide Antigen Derived from the V3 Loop of HIV-1 gp120" @default.
• W2065572647 cites W1531523283 @default.
• W2065572647 cites W1536128836 @default.
• W2065572647 cites W1568767105 @default.
• W2065572647 cites W1964610252 @default.
• W2065572647 cites W1965127482 @default.
• W2065572647 cites W1970988545 @default.
• W2065572647 cites W1980388661 @default.
• W2065572647 cites W1983678802 @default.
• W2065572647 cites W1986736371 @default.
• W2065572647 cites W1993698201 @default.
• W2065572647 cites W1996107940 @default.
• W2065572647 cites W2004360551 @default.
• W2065572647 cites W2008522253 @default.
• W2065572647 cites W2013618384 @default.
• W2065572647 cites W2014333438 @default.
• W2065572647 cites W2019213007 @default.
• W2065572647 cites W2020718845 @default.
• W2065572647 cites W2033684067 @default.
• W2065572647 cites W2042200110 @default.
• W2065572647 cites W2048574795 @default.
• W2065572647 cites W2054476347 @default.
• W2065572647 cites W2069079485 @default.
• W2065572647 cites W2070146818 @default.
• W2065572647 cites W2076584494 @default.
• W2065572647 cites W2079723355 @default.
• W2065572647 cites W2080628353 @default.
• W2065572647 cites W2081992461 @default.
• W2065572647 cites W2089652099 @default.
• W2065572647 cites W2118864235 @default.
• W2065572647 cites W2169227901 @default.
• W2065572647 cites W2231614804 @default.
• W2065572647 cites W4232385589 @default.
• W2065572647 cites W4252432732 @default.
• W2065572647 doi "https://doi.org/10.1074/jbc.272.50.31407" @default.
• W2065572647 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9395472" @default.
• W2065572647 hasPublicationYear "1997" @default.
• W2065572647 type Work @default.
• W2065572647 sameAs 2065572647 @default.
• W2065572647 citedByCount "5" @default.
• W2065572647 crossrefType "journal-article" @default.
• W2065572647 hasAuthorship W2065572647A5014486140 @default.
• W2065572647 hasAuthorship W2065572647A5035433072 @default.
• W2065572647 hasBestOaLocation W20655726471 @default.
• W2065572647 hasConcept C11413529 @default.
• W2065572647 hasConcept C114614502 @default.
• W2065572647 hasConcept C147483822 @default.
• W2065572647 hasConcept C153911025 @default.
• W2065572647 hasConcept C159047783 @default.
• W2065572647 hasConcept C184670325 @default.
• W2065572647 hasConcept C185592680 @default.
• W2065572647 hasConcept C195616568 @default.
• W2065572647 hasConcept C2776235265 @default.
• W2065572647 hasConcept C2779281246 @default.
• W2065572647 hasConcept C2779921545 @default.
• W2065572647 hasConcept C3013748606 @default.
• W2065572647 hasConcept C33923547 @default.
• W2065572647 hasConcept C41008148 @default.
• W2065572647 hasConcept C54355233 @default.
• W2065572647 hasConcept C55493867 @default.
• W2065572647 hasConcept C86803240 @default.
• W2065572647 hasConceptScore W2065572647C11413529 @default.
• W2065572647 hasConceptScore W2065572647C114614502 @default.
• W2065572647 hasConceptScore W2065572647C147483822 @default.
• W2065572647 hasConceptScore W2065572647C153911025 @default.
• W2065572647 hasConceptScore W2065572647C159047783 @default.
• W2065572647 hasConceptScore W2065572647C184670325 @default.
• W2065572647 hasConceptScore W2065572647C185592680 @default.
• W2065572647 hasConceptScore W2065572647C195616568 @default.
• W2065572647 hasConceptScore W2065572647C2776235265 @default.
• W2065572647 hasConceptScore W2065572647C2779281246 @default.
• W2065572647 hasConceptScore W2065572647C2779921545 @default.
• W2065572647 hasConceptScore W2065572647C3013748606 @default.
• W2065572647 hasConceptScore W2065572647C33923547 @default.
• W2065572647 hasConceptScore W2065572647C41008148 @default.
• W2065572647 hasConceptScore W2065572647C54355233 @default.
• W2065572647 hasConceptScore W2065572647C55493867 @default.
• W2065572647 hasConceptScore W2065572647C86803240 @default.
• W2065572647 hasIssue "50" @default.
• W2065572647 hasLocation W20655726471 @default.
• W2065572647 hasOpenAccess W2065572647 @default.
• W2065572647 hasPrimaryLocation W20655726471 @default.
• W2065572647 hasRelatedWork W1487857142 @default.
• W2065572647 hasRelatedWork W1702322095 @default.
• W2065572647 hasRelatedWork W2049696483 @default.
• W2065572647 hasRelatedWork W2080319452 @default.
• W2065572647 hasRelatedWork W2085590670 @default.
• W2065572647 hasRelatedWork W2088670007 @default.
• W2065572647 hasRelatedWork W2163549559 @default.
• W2065572647 hasRelatedWork W2163951709 @default.
• W2065572647 hasRelatedWork W2952230677 @default.
• W2065572647 hasRelatedWork W4200216331 @default.

• next