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H A Scheraga

Publications and source records attributed to H A Scheraga.

At least 19 recordsLinked to original sources
PubMed1992

Equilibrium and kinetic constants for the thiol-disulfide interchange reaction between glutathione and dithiothreitol.

The equilibrium and rate constants for the reaction between oxidized and reduced glutathione and oxidized and reduced dithiothreitol have been determined at several pH values and temperatures. The measurements involve approach to equilibrium from both directions, quenching of the reaction by lowering the pH or by addition of methyl methanethiosulfonate, separation of reactants and products by reverse-phase HPLC, and determination of their concentrations. Analysis of reaction mixtures was carried out at various times to assure that equilibrium had been reached and to determine kinetic constants prior to the attainment of equilibrium.

Disulfides↗
PubMed1992

Role of loop-helix interactions in stabilizing four-helix bundle proteins.

One of the critical issues regarding proteins with a four-helix bundle motif is which interactions play the major role in stabilizing this type of folded structure: the interaction among the four alpha-helices or the interaction between the loop and helix segments. To answer this question, an energetic analysis has been carried out for three proteins with a four-helix bundle--namely, methemerythrin, cytochrome b-562, and cytochrome c'. The structures on which the analysis has been made were derived from their respective crystallographic coordinates. All three proteins have long helices (16-26 residues) and most of their loops are short (3-5 residues). However, it was found in all three proteins that loop-helix interactions were stronger than helix-helix interactions. Moreover, not only the nonbonded component but also the electrostatic component of the interaction energy were dominated by loop-helix interactions rather than by interhelix interactions, although the latter involve favorable helix-dipole interactions due to the antiparallel arrangement of neighboring helices. The results of the energetic analysis indicate that the loop segments, whether they are in a theoretical model or in real proteins, play a significant role in stabilizing proteins with four-helix bundles.

Cytochrome b Group↗
PubMed1992

The intrinsic helix-forming tendency of L-alanine.

Conformational energy calculations have been carried out for three hexadecapeptides in water--namely, a copolymer with the sequence acetyl-AAAAKAAAA-KAAAAKA-amide, 3K(I), in both the charged and neutral forms; a neutral peptide with the sequence acetyl-AAQAAAAQAAAAQAAY-amide, AQY; and a 16-residue L-alanine homopolymer with acetyl and amide terminal groups. The conformational energy was a sum of the empirical conformational energy program for peptides (ECEPP/2) potential energy plus continuum hydration free energy. An empirical (JRF) parameter set was used for the hydration free energy, together with an electrostatic contribution to the solvent effect from charged lysines. The computed relatively high helix content of the most probable conformation of charged 3K(I) and the intermediate helix content of AQY agree reasonably well with experimental values. The computed very low helix content of the alanine homopolymer agrees with experiments on block copolymers and on host-guest random copolymers. The calculations suggest that the high helix content computed for 3K(I) is due to the sum of internal and hydration free energies of the lysine residues rather than to a high intrinsic helix-forming tendency of alanine. The principal component lowering the computed helix contents of AQY and the alanine copolymer relative to 3K(I) is hydration.

Acetylation↗
PubMed1992

Effects of compact volume and chain stiffness on the conformations of native proteins.

An investigation of the statistical properties of the native conformations of proteins, observed from crystal structures, is reported. Protein conformations were analyzed in terms of a bond vector correlation function and molecular volume. It was observed that, while the volume of a protein structure varies nearly linearly with the number of residues, the bond vector correlation function exhibits a universal feature for all sizes of proteins. To interpret the nature of the bond vector correlation function of native protein structures quantitatively, Monte Carlo simulations of realistic polypeptide chains of specific but arbitrary amino acid sequence were carried out. The molecule was constrained in an ellipsoidal volume determined by its chain length, and conformations with unacceptable nonbonded contacts between different amino acid residues were excluded. The interactions within a terminally blocked single residue, which correlate two nearest-neighbor peptide groups in a chain, were taken into account by an energetically biased sampling of its phi-psi space. The simulated chain correlation functions were found to be in good agreement with those of the crystal structures of beta-sheet-type and mixed-type (alpha+beta) proteins of similar length. On the basis of these calculations, it is concluded that the observed conformations of these native proteins may arise from two basic factors: the compactness of structures under hydrophobic interactions and the intrinsic stiffness of polypeptide chains due to the interactions within each terminally blocked residue.

Biophysical Phenomena↗
PubMed1992

Thrombin hydrolysis of an N-terminal peptide from fibrinogen Lille: kinetic and NMR studies.

Fibrinogen Lille, a congenital dysfibrinogenemia, has been reported to arise from a mutation from Asp to Asn at position 7 of the A alpha chain of human fibrinogen, thereby reducing the thrombin-catalyzed rate of hydrolysis of the Arg(16)-Gly(17) peptide bond of this chain. Synthetic peptides of relevant portions of the wild-type and mutant A alpha chains were prepared, and the thrombin-catalyzed rates of hydrolysis of their Arg(16)-Gly(17) peptide bonds were determined. In addition, transferred NOE measurements were made to deduce their conformations, when complexed to bovine thrombin. The kinetics data showed little difference in the hydrolysis rates between the wild-type and mutant peptides, and the NMR data indicate no difference in the bound conformation of these two peptides. Therefore, electrostatic (or salt-bridge) interactions between Asp(7) and thrombin do not influence the bound conformations of these peptides. Asp(7) may interact with a remote residue of fibrinogen, not present in these synthetic peptides, or there may be additional mutations beyond A alpha (1-20) which have not been detected in fibrinogen Lille. Alternatively, when thrombin binds to fibrinogen at its secondary binding site, its primary (active) site may display different reactivities toward wild-type fibrinogen and fibrinogen Lille.

Afibrinogenemia↗
PubMed1992

Solution structure of murine epidermal growth factor determined by NMR spectroscopy and refined by energy minimization with restraints.

The solution structure of murine epidermal growth factor (mEGF) at pH 3.1 and a temperature of 28 degrees C has been determined from NMR data, using distance geometry calculations and restrained energy minimization. The structure determination is based on 730 conformational constraints derived from NMR data, including 644 NOE-derived upper bound distance constraints, constraints on the ranges of 32 dihedral angles based on measurements of vicinal coupling constants, and 54 upper and lower bound constraints associated with nine hydrogen bonds and the three disulfide bonds. The distance geometry interpretation of the NMR data is based on previously published sequence-specific 1H resonance assignments [Montelione et al. (1988) Biochemistry 27, 2235-2243], supplemented here with individual assignments for some side-chain amide, methylene, and isopropyl methyl protons. The molecular architecture of mEGF is the same as that described previously [Montelione et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 5226-5230], but the structure is overall more precisely determined by a more extensive set of NMR constraints. Analysis of proton NMR line widths, amide proton exchange rates, and side-chain 3J(H alpha-H beta) coupling constants provides evidence for internal motion in several regions of the mEGF molecule. Because mEGF is one member of a large family of homologous growth factors and protein domains for which X-ray crystal structures are not yet available, the atomic coordinates resulting from the present structure refinement (which we have deposited in the Brookhaven Protein Data Bank) are important data for understanding the structures of EGF-like proteins and for further detailed comparisons of these structures with mEGF.

Amino Acid Sequence↗
PubMed1992

Empirical solvation models in the context of conformational energy searches: application to bovine pancreatic trypsin inhibitor.

Continuum solvation models that estimate free energies of solvation as a function of solvent accessible surface area are computationally simple enough to be useful for predicting protein conformation. The behavior of three such solvation models has been examined by applying them to the minimization of the conformational energy of bovine pancreatic trypsin inhibitor. The models differ only with regard to how the constants of proportionality between free energy and surface area were derived. Each model was derived by fitting to experimentally measured equilibrium solution properties. For two models, the solution property was free energy of hydration. For the third, the property was NMR coupling constants. The purpose of this study is to determine the effect of applying these solvation models to the nonequilibrium conformations of a protein arising in the course of global searches for conformational energy minima. Two approaches were used: (1) local energy minimization of an ensemble of conformations similar to the equilibrium conformation and (2) global search trajectories using Monte Carlo plus minimization starting from a single conformation similar to the equilibrium conformation. For the two models derived from free energy measurements, it was found that both the global searches and local minimizations yielded conformations more similar to the X-ray crystallographic structures than did searches or local minimizations carried out in the absence of a solvation component of the conformational energy. The model derived from NMR coupling constants behaved similarly to the other models in the context of a global search trajectory. For one of the models derived from measured free energies of hydration, it was found that minimization of an ensemble of near-equilibrium conformations yielded a new ensemble in which the conformation most similar to the X-ray determined structure PTI4 had the lowest total free energy. Despite the simplicity of the continuum solvation models, the final conformation generated in the trajectories for each of the models exhibited some of the characteristics that have been reported for conformations obtained from molecular dynamics simulations in the presence of a bath of explicit water molecules. They have smaller root mean square (rms) deviations from the experimentally determined conformation, fewer incorrect hydrogen bonds, and slightly larger radii of gyration than do conformations derived from search trajectories carried out in the absence of solvent.

Animals↗
PubMed1991

Fluorescence resonance energy transfer mapping of the fourth of six nucleotide-binding sites of chloroplast coupling factor 1.

Equilibrium dialysis measurements of adenine nucleotide binding to chloroplast coupling factor 1 suggest that the enzyme has six binding sites for ADP, adenylyl-beta,gamma-imidodiphosphate (AMP-PNP), and 2'(3')-O-2,4,6-trinitrophenyl-ATP (TNP-ATP). High affinity binding at all six sites requires the divalent cation, Mg2+. Three of the nucleotide-binding sites, sites 1, 2, and 3, have been mapped by fluorescence resonance energy transfer distance measurements (see McCarty, R. E., and Hammes, G. G. (1987) Trends Biochem. Sci. 12, 234-237). Using the same technique, we mapped the location of nucleotide-binding site 4, a tight, exchangeable site (Shapiro, A. B., Huber, A. H., and McCarty, R. E. (1991) J. Biol. Chem. 266, 4194-4200). Two arrangements of the energy transfer map of coupling factor 1 were found which are compatible with the available data. The two arrangements make different predictions about which sites interact in cooperative catalysis.

Adenosine Diphosphate↗
PubMed1991

Conformational studies of a peptide corresponding to a region of the C-terminus of ribonuclease A: implications as a potential chain-folding initiation site.

Conformational properties of the OT-16 peptide, the C-terminal 20 amino acids of RNase A, were examined by nonradiative energy transfer. A modified OT-16 peptide was prepared by solid-phase synthesis with the inclusion of diaminobutyric acid (DABA) at the C-terminus. The OT-16-DABA peptide was labeled with a fluorescent 1,5-dimethylaminonaphthalene sulfonyl (dansyl, DNS) acceptor at the N-terminal amine and a fluorescent naphthoxyacetic acid (NAA) donor at the gamma-amine of the DABA located at the C-terminus of the peptide by using an orthogonal protection scheme. Energy transfer was monitored in DNS-OT-16-DABA-NAA by using both fluorescence intensity (sensitized emission) and lifetime (donor quenching) experiments. The lifetime data indicate that the peptide system is a dynamic, flexible one. A detailed analysis, based on a dynamic model that includes a skewed Gaussian function to model the equilibrium distribution of interprobe distances and a mutual diffusion coefficient between the two probes to model conformational dynamics in the peptide [Beechem & Haas (1989) Biophys. J. 55, 1225.], identified the existence of a partially ordered structure (relatively narrow distribution of interprobe distances) at temperatures greater than or equal to 20 degrees C in the absence of denaturant. The width and the position of the average of the distributions decrease with increasing temperature, in this range; this suggests that the structure is stabilized by hydrophobic interactions. In addition, the peptide undergoes cold denaturation at around 1.5 degrees C as indicated by broadening of the distance distribution. The addition of 6 M guanidine hydrochloride (Gdn-HCl) also broadens the distance distribution significantly, presumably by eliminating the hydrophobic interactions and unfolding the peptide. The results of the analysis of the distance distribution demonstrate that (1) nonradiative energy transfer can be used to study the conformational dynamics of peptides on the nanosecond time scale, (2) a partially ordered structure of OT-16-DABA exists in solution under typical refolding conditions, and (3) structural constraints (presumably hydrophobic interactions) necessary for the formation of a chain-folding initiation site in RNase A are also present in the OT-16-DABA peptide in the absence of denaturant and are disrupted by Gdn-HCl.

Amino Acid Sequence↗
PubMed1991

Calculation of protein conformation as an assembly of stable overlapping segments: application to bovine pancreatic trypsin inhibitor.

Conformations of bovine pancreatic trypsin inhibitor were calculated by assuming that the final structure as well as properly chosen overlapping segments thereof are simultaneously in low-energy (not necessarily the lowest-energy) conformational states. Therefore, the whole chain can be built up from building blocks whose conformations are determined primarily by short-range interactions. Our earlier buildup procedure was modified by taking account of a statistical analysis of known amino acid sequences that indicates that there is nonrandom pairing of amino acid residues in short segments along the chain, and by carrying out energy minimization on only these segments and on the whole chain [without minimizing the energies of intermediate-size segments (20-30 residues long)]. Results of this statistical analysis were used to determine the variable sizes of the overlapping oligopeptide building blocks used in the calculations; these varied from tripeptides to octapeptides, depending on the amino acid sequence. Successive stages of approximations were used to combine the low-energy conformations of these building blocks in order to keep the number of variables in the computations to a manageable size. The calculations led to a limited number of conformations of the protein (only two different groups, with very similar structure within each group), most residues of which were in the same conformational state as in the native structure.

Amino Acid Sequence↗
PubMed1991

The electrostatically driven Monte Carlo method: application to conformational analysis of decaglycine.

The Electrostatically Driven Monte Carlo (EDMC) method was applied in a study of a decamer of glycine whose conformational behavior is described by the Empirical Conformational Energy Program for Peptides (ECEPP/2) potential energy model. When free neutral end groups were used, it was found that conformations that were not alpha-helical had significantly lower potential energies than fully alpha-helical ones. However, when the N- and C-termini were blocked by acetyl and methyl amide groups, respectively, the number of unsatisfied hydrogen-bond donors and acceptors at the helix termini was diminished from 8 to 6; in this case, the possibility of forming two additional alpha-helical hydrogen bonds was an important enough factor in making the alpha-helical conformation the one with the lowest energy. The EDMC method was used as a global energy optimizer since it does not often become trapped in high-energy local minima.

Electrochemistry↗
PubMed1991

U-3'-BCIP: a chromogenic substrate for the detection of RNase A in recombinant DNA expression systems.

The synthesis of the bovine pancreatic ribonuclease A (RNase A, EC 3.1.27.5) chromogenic substrate uridine-3'-(5-bromo-4-chloroindol-3-yl)-phosphate (U-3'-BCIP) is described. RNase A catalyzes the hydrolysis of U-3'-BCIP to release a halogenated indol-3-ol that undergoes rapid aerobic oxidation to the dark blue 5,5'-dibromo-4,4'-dichloroindigo. Preliminary kinetic studies indicate that this compound may have practical use for assaying RNase A activity both in vitro and in vivo, e.g. in screening bacterial colonies for RNase A produced by recombinant DNA methods.

Animals↗
PubMed1991

Conformational energy studies of beta-sheets of model silk fibroin peptides. I. Sheets of poly(Ala-Gly) chains.

A new model structure is proposed for the silk I form of the crystalline domains of Bombyx mori silk fibroin and the corresponding crystal form of poly(L-Ala-Gly). It was deduced from conformational energy computations on stacked sheet structures of poly(L-Ala-Gly). The novel sheet structure contains interstrand hydrogen bonds but is composed of anti-parallel polypeptide chains whose conformation differs from that of the antiparallel beta-sheets that constitute the silk II structure. The strands of the new sheet have a two-residue repeat, in which the Ala residues adopt a right-handed and the Gly residues a left-handed sheet-like conformation. The computed unit cell is orthorhombic, with cell dimensions a = 8.94 A, b = 6.46 A, and c = 11.26 A. The model accounts for most spacings in the observed fiber x-ray diffraction patterns of silk I and of the silk-I-like form of poly(L-Ala-Gly), and it is consistent with nmr and ir spectroscopic data. As a test of the computations, the well-established beta-sheet structure of silk II and the corresponding form of poly(L-Ala-Gly) have been reproduced. The computed energies for the two forms of poly(L-Ala-Gly) indicate that the silk-II-like form is more stable, by about 1.0 kcal/mol per residue. The main difference between the two structures is the orientation of the Ala side chains of neighboring strands in each sheet. In the Pauling-Corey beta-sheet and in the silk II form, referred to as an "in-register" structure, the Ala side chains of every strand point to the same side of a sheet. In the silk I structure, referred to as "out-of-register," the side chains of Ala residues in adjacent strands point to opposite sides of the sheet.

Amino Acid Sequence↗
PubMed1991

On the multiple-minima problem in the conformational analysis of polypeptides. V. Application of the self-consistent electrostatic field and the electrostatically driven Monte Carlo methods to bovine pancreatic trypsin inhibitor.

In connection with the accompanying paper to test various models for the hydration of polypeptides, we have explored a limited portion of the conformational energy hyperspace of the small protein bovine pancreatic trypsin inhibitor (BPTI) with the aid of two search methods developed in this laboratory. A series of low-energy conformations was obtained as a result of this study. These conformations constitute a set of local minima in the conformational energy space of the molecule as described by the ECEPP/2 (Empirical Conformational Energy Program for Peptides) potential energy function, without the inclusion of hydration. Five different initial conformations were used in this exploration: the first corresponds to an energy-refined structure based on the crystallographic coordinates (4PTI) provided by Deisenhofer and Steigemann and reported previously by Meirovitch and Scheraga. The remaining four initial conformations were obtained by using a Variable-Target-Function procedure, applied to the experimental Cartesian coordinates (5PTI) reported by Wlodawer et al. The self-consistent electrostatic field (SCEF) and the electrostatically driven Monte Carlo (EDMC) methods were used to search the conformational space. The SCEF and EDMC methodologies assume that a polypeptide or protein molecule is driven toward the native structure mainly by the action of the electrostatic interactions. Application of these methodologies led to a set of conformations (up to 50 kcal/mol lower than the starting ones) with ECEPP/2 energies lower than any of those that we had previously found. Application of both methods to the initial conformation generated from 4PTI led to a series of low-energy conformations exhibiting similar rms deviations with respect to the experimental data (4PTI) as did the starting conformation. However, statistical analysis of the runs that had started from the conformations generated by using the variable-target-function procedure (and applying the EDMC method) indicated that the rms deviations of the atomic positions of the new low-energy conformations tended to increase as the energy improved, when compared with the X-ray data from which the starting conformations had been generated. The structures with the lowest energies also had radii of gyration smaller than the experimentally observed one. These results indicated a need to include hydration in the potential function, and provided the conformations used in the accompanying paper to test various hydration models.

Animals↗
PubMed1991

Empirical solvation models can be used to differentiate native from near-native conformations of bovine pancreatic trypsin inhibitor.

Several hydration models for peptides and proteins based on solvent accessible surface area have been proposed previously. We have evaluated some of these models as well as four new ones in the context of near-native conformations of a protein. In addition, we propose an empirical site-site distance-dependent correction that can be used in conjunction with any of these models. The set of near-native structures consisted of 39 conformations of bovine pancreatic trypsin inhibitor (BPTI) each of which was a local minimum of an empirical energy function (ECEPP) in the absence of solvent. Root-mean-square (rms) deviations from the crystallographically determined structure were in the following ranges: 1.06-1.94 A for all heavy atoms, 0.77-1.36 A for all backbone heavy atoms, 0.68-1.33 A for all alpha-carbon atoms, and 1.41-2.72 A for all side-chain heavy atoms. We have found that there is considerable variation among the solvent models when evaluated in terms of concordance between the solvation free energy and the rms deviations from the crystallographically determined conformation. The solvation model for which the best concordance (0.939) with the rms deviations of the C alpha atoms was found was derived from NMR coupling constants of peptides in water combined with an exponential site-site distance dependence of the potential of mean force. Our results indicate that solvation free energy parameters derived from nonpeptide free energies of hydration may not be transferrable to peptides. Parameters derived from peptide and protein data may be more applicable to conformational analysis of proteins. A general approach to derive parameters for free energy of hydration from ensemble-averaged properties of peptides in solution is described.

Animals↗
PubMed1991

Spectroscopic, immunochemical, and thermodynamic properties of carboxymethyl(Cys6, Cys127)-hen egg white lysozyme.

A three-disulfide form of hen egg white lysozyme with Cys6 and Cys127 blocked by carboxymethyl groups was prepared, purified, and characterized for eventual use in protein folding experiments. Trypsin digestion followed by proline-specific endopeptidase digestion facilitated the unambiguous assignment of the disulfide bond pairings and the modified residues in this derivative. 3SS-lysozyme demonstrated nearly full enzymatic activity at its pH optimum, pH 5.5. The 3SS-lysozyme derivative and unmodified lysozyme were shown to be identical by CD spectroscopy at pH 3.6. Immunochemical binding assays demonstrated that the conformation of lysozyme was perturbed predominantly only locally by breaking and blocking the disulfide bond between Cys6 and Cys127. Both 3SS-lysozyme and unmodified lysozyme exhibited reversible thermally induced transitions at pH 2.0, but the Tm of 3SS-lysozyme, 18.9 degrees C, was found to be 34 degrees lower than that of native lysozyme under the same conditions. The conformational chemical potential of the denatured form of unmodified lysozyme was determined from the transition curves to be approximately 6.7 kcal/mol higher than that of the denatured form of 3SS-lysozyme, at pH 2.0 and 35 degrees C, if the conformational chemical potential for the folded forms of both 3SS-lysozyme and unmodified lysozyme is arbitrarily assumed to be 0.0 kcal/mol. A calculation of the increase in the theoretical loop entropy of denatured 3SS-lysozyme resulting from the cleavage of the Cys6-Cys127 disulfide bond, however, yielded a value of only 5.4 kcal/mol for the difference in conformational chemical potential. This suggests that, in addition to the entropic component, there is also an enthalpic contribution to the difference in the conformational chemical potential corresponding to approximately 1.3 kcal/mol. Thus, it is concluded that the reduction and blocking of the disulfide bond between Cys6 and Cys127 destabilizes 3SS-lysozyme relative to unmodified lysozyme predominantly by stabilizing the denatured conformation by increasing its chain entropy.

Chromatography, High Pressure Liquid↗
PubMed1991

Investigation of a physical basis for conformational similarity in proteins.

Amino acid residues in a globular protein fold against one another into a compact structure. We have sought common physical factors within similarly folded backbone structures in such proteins which might influence the folding and which could be used in predicting the backbone structure. The physical factors examined are the 10 orthogonal ones identified by Kidera et al. (1985a). Comparison of the smoothed physical factor profiles between sequences, which have similar backbone structures, shows that there is good agreement among the profiles of helical stretches, but not for other backbone structures that have been examined. This is ascribed to the fact that helical structures involve local interactions, which then require similar physical profiles to form, but that other structures are not so strongly locally determined in the native structure.

Amino Acid Sequence↗