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K Ramnarayan

Publications and source records attributed to K Ramnarayan.

At least 19 recordsLinked to original sources
PubMed1997

Analysis of high-affinity binding determinants in the receptor binding epitope of basic fibroblast growth factor.

Basic fibroblast growth factor (bFGF) is implicated in the pathogenesis of several vascular and connective diseases. A key step in the discovery of bFGF receptor antagonists to mitigate these actions is to define the functional epitope required for receptor binding of the growth factor. In previous studies, we identified Glu96 as an essential residue in this epitope using site-directed mutagenesis. Here we examined the role of solvent accessible neighboring residues of Glu96 of bFGF on receptor binding affinity. Wild-type bFGF and its muteins were cloned and expressed in Escherichia coli and evaluated for FGF receptor binding affinity. Replacement of Asn104 of bFGF by alanine reduced receptor binding affinity over 400-fold compared with wild-type bFGF. We next explored the effect of neighboring residues of Asn104 on receptor binding affinity-Muteins in which Arg97, Leu98, Glu99, Asn101, Asn102, Thr105 and Pro141 were individually replaced by alanine exhibited receptor binding similar to wild-type bFGF. By contrast, substitution of Tyr103 or Leu140 by alanine reduced receptor binding affinity about 400- and 150-fold, respectively, in accord with a previous report. We conclude that at least six solvent-accessible residues in bFGF are crucial for high-affinity receptor binding, as evidenced by at least a 10-fold diminution in the affinity of the corresponding alanine muteins. The polar residues Glu96 and Asn104 appear to form an area important for facilitating the initial contact between ligand and receptor, whereas Tyr24, Tyr103, Leu140 and Met142 form a hydrophobic patch that may stabilize the complex. The detailed structure of this functional epitope can be employed in the discovery and design of bFGF antagonists using computational methods.

Asparagine↗
PubMed1995

Glu-96 of basic fibroblast growth factor is essential for high affinity receptor binding. Identification by structure-based site-directed mutagenesis.

The importance of basic fibroblast growth factor (bFGF) in several pathophysiological processes has stimulated interest in the design of receptor antagonists to mitigate such effects. Of key importance in this connection is the characterization of the functional binding epitopes of the growth factor for its receptor. Based on peptide mapping and molecular dynamics calculations of the three-dimensional structure of basic fibroblast growth factor, we employed site-directed mutagenesis to investigate the effect of altering residues at positions 107, 109-114, and 96 on bFGF on receptor binding affinity. All muteins were cloned and expressed in Escherichia coli, purified to homogeneity employing heparin-Sepharose columns, and evaluated for receptor binding affinity. We found that replacement of residues at positions 107 and 109-114 by alanine or phenylalanine had little effect on receptor binding affinities compared with wild type bFGF, in agreement with previous evidence that bFGF residues 109-114 comprise a low affinity binding site. By contrast, substitution of Glu-96 with alanine yielded a molecule having about 0.1% of the affinity of the wild type bFGF. The affinity of the corresponding lysine and glutamine muteins was 0.3 and 10%, respectively, emphasizing the importance of a negative charge at this position. Our findings are consistent with the view that residues 106-115 on bFGF represent a low affinity binding site on bFGF. In addition, we identify Glu-96 as a crucial residue for binding to fibroblast growth factor receptor-1.

Amino Acid Sequence↗
PubMed1995

Conformational studies on model dipeptides of Gly, L-Ala and their C alpha-substituted analogs.

As a part of the development of conformational guidelines for the design of metabolically altered peptidomimetics, we present conformational energy calculations on model dipeptide compounds with glycine (Gly), L-alanine (Ala), alpha-aminoisobutyric acid (Aib), L-tert-butylglycine (Tle), L-phenylglycine (Phg), (alpha, alpha)-diphenylglycine (D phi g), L-2-aminobutyric acid (Abu), 2-amino-2-ethylbutyric acid (Deg), L-2-amino-2-vinylacetic acid (Ava) and (alpha, alpha)-divinylglycine (Dvg). The energy calculations have been made using molecular mechanics methods with a force field derived from MM2. The salient features are expressed in terms of conformational energy plots, drawn as a function of the backbone torsion angles phi(Ci'-1-Ni-Ci alpha i-Ci') and psi(Ni-Ci alpha -C'-N(i+1)). The low-energy structures of these compounds are qualitatively consistent with the X-ray crystal structure analyses of peptides and peptidomimetics. They are also in agreement with the results of the solution-phase studies carried out by NMR and IR techniques. The results obtained have important implications in the design of conformationally restricted peptidomimetics.

Alanine↗
PubMed1995

Antibody humanization predicted by computer graphic analysis.

Humanized antibodies continue to be evaluated in clinical trials, and each trial will yield valuable information for the design of the next generation of humanized antibodies. More attention will be given to the use of antibody fragments and the selection of human antibody specificities from phage libraries for human antibody therapeutics. Computer graphic analysis and molecular modeling of antibody structures will continue to be invaluable for predicting structural modifications for humanization of antibodies and for extracting structural information from antigen binding sites that can ultimately lead to small molecule therapeutics.

Animals↗
PubMed1993

Accessing three-dimensional chemical information in antibody molecules.

Important structural information can be obtained from monoclonal antibodies through X-ray crystallographic analysis and computer modeling of CDR loops. These applications of monoclonal antibody technology offer new insights and novel approaches to the modification of antibody molecules for disease treatment, diagnosis, and drug design.

Antibodies, Monoclonal↗
PubMed1991

Identification of an extracytoplasmic region of H+,K(+)-ATPase labeled by a K(+)-competitive photoaffinity inhibitor.

The photoaffinity reagent 8-[(4-azidophenyl)-methoxy]-1-tritiomethyl-2, 3-dimethylimidazo-[1,2-alpha]pyridinium iodide ([3H]mDAZIP) has been synthesized and used to photoinactivate and label purified hog gastric H+,K(+)-ATPase. The specific (K(+)-sensitive) components of both photoinactivation and labeling showed dependences on inhibitor concentration consistent with covalent modification at an extracytoplasmic site of reversible K(+)-competitive binding in the dark. The maximum amount of specific labeling (1.2 nmol/mg) was similar to the number of phosphorylation sites measured (1.0 +/- 0.14 nmol/mg). Specific labeling was distributed 76% on the alpha chain, 18% on the beta chain, and 6% on undefined peptides. Various digestions with trypsin, protease V8, and thermolysin were employed to fragment the labeled enzyme. Gasphase sequencing of the radioactive peptides identified the major site of specific labeling to be within a region where only two stretches of amino acids (Leu105 to Ile126 and Leu139 to Phe155, designated H1 and H2, respectively) are predicted to span the membrane. This in turn suggested that the labeling site was located within or close to the proposed loop between them (Gln127 to Asn138). A computer-driven energy minimization protocol yielded a loop structure to which SCH 28080 (the parent structure of [3H]mDAZIP) could be docked. Conversely, modeling of the corresponding region of Na+,K(+)-ATPase (a homologous enzyme with much lower affinity for SCH 28080) yielded no apparent binding site. Similarities in the inhibition of H+,K(+)-ATPase by SCH 28080 and of Na+,K(+)-ATPase by ouabain lead to the hypothesis that, in each case, inhibitor binding to E2-P is associated with an increase in the hydrophobicity of the environment of the loop between H1 and H2.

Adenosine Triphosphatases↗
PubMed1985

Peripheral ameloblastoma.

The clinical presentation and histological features of peripheral ameloblastoma, occurring on the floor of the mouth in a 65-year-old male, are described. The lesion was surgically excised. All the classical features of peripheral ameloblastoma were seen in this patient.

Aged↗