PubMed Health⌕ Search

Biomedical subjects

E O Purisima

Publications and source records attributed to E O Purisima.

10 recordsLinked to original sources
PubMed1996

Nonpolar interactions of thrombin and its inhibitors at the fibrinogen recognition exosite: thermodynamic analysis.

Nonpolar interactions play a major role in the association of the fibrinogen recognition exosite of thrombin with the C-terminal fragment (55-65), Asp-Phe-Glu-IIe-Pro-Glu-Glu-Tyr-Leu-Gln, of hirudin, which is a naturally occurring thrombin inhibitor. The thermodynamic details (free energy, enthalpy, entropy, and heat capacity) of the molecular recognition are studied by using five analogs of a synthetic bivalent thrombin inhibitor (P552), tert-butylbenzensulfonyl-Arg-(D-pipecolic acid)-(12-amino-dodecanoic acid)-(gamma-aminobutyric acid)-hirudin55-65. The residue of PheH56, IleH59, ProH60, TyrH63, or LeuH64 in hirudin 55-65 segment is substituted by Gly in each analog in order to elucidate the contributions of these nonpolar side chains. The results show that the interactions of these nonpolar side chains with thrombin are enthalpy-driven, except for the contribution of the PheH56 side chain which is entropy-driven. Interestingly, molecular modeling predicts a large conformational change due to the Gly substitution of PheH56. In analyzing the correlation among the thermodynamic and structural properties of the nonpolar interaction, a good correlation is observed between the binding free energy and the hydrophobicity of the molecular surface; i.e., tighter binding is observed as more nonpolar atoms are buried and more polar atoms are exposed upon molecular association.

Antithrombins↗
PubMed1995

Calculation of relative binding free energies and configurational entropies: a structural and thermodynamic analysis of the nature of non-polar binding of thrombin inhibitors based on hirudin55-65.

Free energy calculations were carried out on a series of exosite-binding inhibitors of thrombin. These inhibitors are based on the C-terminal fragment of hirudin and have the sequence Phe-Glu-Glu-IleH59-Pro-Glu-Glu-Tyr- Leu, where the superscript over Ile indicates its relative position in the natural sequence of hirudin. In this study, the effect of replacing IleH59 with ten other non-polar amino acids was examined. Three preferred interaction sites for methyl/methylene groups for the various XaaH59 side-chains in the complex were identified from conformational search calculations. The corresponding thermodynamic changes were determined using a combination of systematic search and energy minimization in a manner that locates the local minima in the system and in the process simultaneously builds up the partition function. The free energy, internal energy and entropic contributions are readily calculated from the partition function. Very good agreement in the resulting relative binding free energies was obtained between theory and experiment. The calculations allowed us to dissect out the enthalpic, entropic and solvation contributions to delta delta G. The contribution from desolvation was found to be relatively weak. The binding of these non-polar side-chains to thrombin is found to be driven mainly by favorable protein-ligand interactions rather than by the desire for non-polar groups to be desolvated. We also find that the configurational entropy contributes about 0.48 kcal/mol (0.81 kappa T) in average for each torsional angle "frozen" in binding.

Algorithms↗
PubMed1992

Conformationally restricted thrombin inhibitors resistant to proteolytic digestion.

A new type of thrombin exo-site inhibitor has been designed with enhanced inhibitory potency and increased metabolic stability. With the aid of the model of the structure of the thrombin-hirudin fragment complex [Yue, S.-Y., DiMaio, J., Szewczuk, Z., Purisima, E. O., Ni, F., & Konishi, Y. (1992) Protein Eng. 5, 77-85], cyclic analogs of the hirudin fragment (hirudin55-65) were designed and synthesized. In these analogs, the side chains of appropriately substituted residues, 58 and 61, were joined in order to restrict the conformation of the inhibitor. An analog with an 18-membered lactam ring showed higher antithrombin activity (IC50 = 0.57 microM) than the corresponding analogs with 17- or 16-membered rings and was 2-fold more potent than its linear counterpart. Even 4-fold greater enhancement was obtained when a shorter fragment, hirudin 55-62, was cyclized. This cyclization not only improved the potency but, more importantly, dramatically increased the resistance to proteolytic digestion. Remarkable enhancement of stability to proteolysis was observed for peptide bonds located in the exocyclic linear peptide segments. These results are discussed using molecular modeling.

Amino Acid Sequence↗
PubMed1992

Conformational stability of a thrombin-binding peptide derived from the hirudin C-terminus.

The COOH-terminal region of hirudin represents an independent functional domain that binds to an anion-binding exosite of thrombin and inhibits the interaction of thrombin with fibrinogen and regulatory proteins in blood coagulation. The thrombin-bound structure of the peptide fragment, hirudin 55-65, has been determined by use of transferred NOE spectroscopy [Ni, F., Konishi, Y., & Scheraga, H. A. (1990) Biochemistry 29, 4479-4489]. The stability of the thrombin-bound conformation has been characterized further by a combined NMR and theoretical analysis of the conformational ensemble accessible by the hirudin peptide. Medium- and long-range NOE's were found for the free hirudin peptide in aqueous solution and in a mixture of dimethyl sulfoxide and water at both ambient (25 degrees C) and low (0 degrees C) temperatures, suggesting that ordered conformations are highly populated in solution. The global folding of these conformations is similar to that in the thrombin-bound state, as indicated by NOE's involving the side-chain protons of residues Phe(56), Ile(59), Pro(60), Tyr(63), and Leu(64). Residues Glu(61), Glu(62), Tyr(63), and Leu(64) all contain approximately 50% of helical conformations calculated from the ratio of the sequential dNN and d alpha N NOE's. Among the helical ensemble, active 3(10)-helical conformations were found by an analysis of the medium-range [(i,i+2) and (i,i+3)] NOE's involving the last six residues of the peptide. An analysis of the side-chain rotamers revealed that, upon binding to thrombin, there may be a rotation around the alpha CH-beta CH bond of Ile(59) such that Ile(59) adopts a gauche- (chi 1 = +60) conformation in contrast to the highly populated trans (chi 1 = -60) found for Ile(59) in the free peptide. However, the thrombin-bound conformation of the hirudin peptide is still an intrinsically stable conformer, and the preferred conformational ensemble of the peptide contains a large population of the active conformation. The apparent preference for a gauche- (chi 1 = +60) side-chain conformation of Ile(59) in the bound state may be explained by the existence of a positively charged arginine residue among the hydrophobic residues in the thrombin exosite.

Binding Sites↗
PubMed1992

Characterization of the interactions of a bifunctional inhibitor with alpha-thrombin by molecular modelling and peptide synthesis.

A potent thrombin inhibitor, [D-Phe45, Arg47] hirudin 45-65, that contains an active site-directed sequence D-Phe-Pro-Arg-Pro, an exosite specific fragment hirudin 55-65 (H55-65) and a linker portion hirudin 49-54, was designed based on the hirudin sequence [DiMaio et al. (1990) J. Biol. Chem., 265, 21698-21798]. A three-dimensional model of the complex between the B-chain of human thrombin and the inhibitor [D-Phe45, Arg47] hirudin 45-65 was constructed using molecular modelling starting from the X-ray C alpha coordinates of the thrombin-hirudin complex and the NMR-derived structure of the thrombin-bound hirudin 55-65. The contribution of the H49-54 fragment to the thrombin-inhibitor interaction was deduced by examining a series of analogs containing single glycine substitution and analogs with reduced number of residues within the linker. The results were consistent with the molecular modelling observations i.e. the H49-54 fragment serves the role of a spacer in the binding interaction and could be replaced by four glycine residues. The studies on the interaction of the exosite-directed portion of the inhibitor with thrombin using a series of synthetic H55-65 analogs demonstrated that residues AspH55 to ProH60 play a major role in binding to human thrombin where the side chains of PheH56, IleH59 and GluH57 showed critical contributions. Molecular modelling suggested that these side chains may contribute to inter- and intramolecular hydrophobic and electrostatic interactions, respectively.

Amino Acid Sequence↗
PubMed1989

Proton NMR assignments and regular backbone structure of bovine pancreatic ribonuclease A in aqueous solution.

Proton NMR assignments have been made for 121 of the 124 residues of bovine pancreatic ribonuclease A (RNase A). During the first stage of assignment, COSY and relayed COSY data were used to identify 40 amino acid spin systems belonging to alanine, valine, threonine, isoleucine, and serine residues. Approximately 60 other NH-alpha CH-beta CH systems were also identified but not assigned to specific amino acid type. NOESY data then were used to connect sequentially neighboring spin systems; approximately 475 of the possible 700 resonances in RNase A were assigned in this way. Our assignments agree with those for 20 residues assigned previously [Hahn, U., & Rüterjans, H. (1985) Eur. J. Biochem. 152, 481-491]. Additional NOESY correlations were used to identify regular backbone structure elements in RNase A, which are very similar to those observed in X-ray crystallographic studies [Wlodawer, A., Borkakoti, N., Moss, D. S., & Howlin, B. (1986) Acta Crystallogr. B42, 379-387].

Amino Acid Sequence↗
PubMed1987

An approach to the multiple-minima problem in protein folding by relaxing dimensionality. Tests on enkephalin.

An algorithm for locating the region in conformational space containing the global energy minimum of a polypeptide is described. Distances are used as the primary variables in the minimization of an objective function that incorporates both energetic and distance-geometric terms. The latter are obtained from geometry and energy functions, rather than nuclear magnetic resonance experiments, although the algorithm can incorporate distances from nuclear magnetic resonance data if desired. The polypeptide is generated originally in a space of high dimensionality. This has two important consequences. First, all interatomic distances are initially at their energetically most favorable values; i.e. the polypeptide is initially at a global minimum-energy conformation, albeit a high-dimensional one. Second, the relaxation of dimensionality constraints in the early stages of the minimization removes many potential energy barriers that exist in three dimensions, thereby allowing a means of escaping from three-dimensional local minima. These features are used in an algorithm that produces short trajectories of three-dimensional minimum-energy conformations. A conformation in the trajectory is generated by allowing the previous conformation in the trajectory to evolve in a high-dimensional space before returning to three dimensions. The resulting three-dimensional structure is taken to be the next conformation in the trajectory, and the process is iterated. This sequence of conformations results in a limited but efficient sampling of conformational space. Results for test calculations on Met-enkephalin, a pentapeptide with the amino acid sequence H-Tyr-Gly-Gly-Phe-Met-OH, are presented. A tight cluster of conformations (in three-dimensional space) is found with ECEPP energies (Empirical Conformational Energy Program for Peptides) lower than any previously reported. This cluster of conformations defines a region in conformational space in which the global-minimum-energy conformation of enkephalin appears to lie.

Algorithms↗
PubMed1986

An approach to the multiple-minima problem by relaxing dimensionality.

A method is presented for starting from a very-low-energy high-dimensional conformation and obtaining a low-energy three-dimensional structure by gradual contraction of the dimensionality. The contraction in dimensionality is achieved by use of Cayley-Menger determinants, of which a simplified form is derived here. Preliminary results are presented for a virtual-bond pentapeptide and for full-atom representations of several terminally blocked amino acids.

Mathematics↗