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M S Doscher

Publications and source records attributed to M S Doscher.

10 recordsLinked to original sources
PubMed1992

Structural changes that accompany the reduced catalytic efficiency of two semisynthetic ribonuclease analogs.

The structures of two catalytically defective semi-synthetic RNases obtained by replacing aspartic acid 121 with asparagine or alanine have been determined and refined at a resolution of 2.0 A (R = 0.186 and 0.172, respectively). When these structures are compared with the refined 1.8-A structure (R = 0.204) of the fully active aspartic acid-containing enzyme (Martin, P.D., Doscher, M.S., and Edwards, B. F. P. (1987) J. Biol. Chem. 262, 15930-15938), numerous and widespread changes, much greater in number and magnitude than the small structural variations noted previously between the semisynthetic complex and RNase A, are found to have occurred. These changes include the movement of the loop containing residues 65-72 away from the active site, a more or less generalized relocation of crystallographically bound water molecules, and a number of rearrangements in the hydrogen bonding network at the active site. Most changes are far removed from the immediate site of the modifications and are distributed essentially throughout the molecule. The details of many of these changes are unique to each analog. In the asparagine analog, a destabilization in the positioning of active site residue His-119 also appears to have occurred.

Alanine↗
PubMed1991

Histidine pKa shifts accompanying the inactivating Asp121----Asn substitution in a semisynthetic bovine pancreatic ribonuclease.

A semisynthetic RNase, RNase-(1-118).(111-124), consisting of a noncovalent complex between residues 1-118 of RNase (obtained from the proteolytic digestion of RNase A), and a synthetic 14-residue peptide containing residues 111-124 of RNase, exhibits 98% of the enzymatic activity of bovine pancreatic ribonuclease A (EC 3.1.27.5). The replacement of aspartic acid-121 by asparagine in this semisynthetic RNase to form the "D121N" analog reduces kcat/Km to 2.7% of the value for RNase A. In the present work, 1H NMR spectroscopy has been used to probe the ionization states of His12, His105, and His119 in this catalytically defective semisynthetic RNase. A comparison of the observed resonances of D121N with those previously determined by others for RNase A enabled us to assign the C2 proton NMR resonances to individual residues; the assignment of His119 was confirmed by titrating D121N with the fully deuterated peptide, [Asn121]-RNase-(111-124). The observed pKa values of His12, His105, and His119 decrease 0.18, 0.16, and 0.02 pH unit, respectively, as a result of the D121N replacement. Values calculated by using a finite difference algorithm to solve the Poisson-Boltzmann equation (the DELPHI program, version 3.0) and a refined 2.0-A coordinate set for the crystal structure of D121N differ significantly for active site residues His12 (delta pKa = -0.58) and His119 (delta pKa = -0.55) but not for His105 (delta pKa = -0.10). The elmination of bound water from the calculations reduced, but did not reconcile, these discrepancies (His12, delta pKa = -0.36; His119, delta pKa = -0.41).

Animals↗
PubMed1987

The refined crystal structure of a fully active semisynthetic ribonuclease at 1.8-A resolution.

A fully active, semisynthetic analog of bovine ribonuclease A, comprised of residues 1-118 of the molecule in a noncovalent complex with the synthetic peptide analog of residues 111-124, has been crystallized in space group P3(2)21 from a solution of 1.3 M ammonium sulfate and 3.0 M cesium chloride at pH 5.2. The crystallographic structure was determined by rotation and translation searches utilizing the coordinates for ribonuclease A reported by Wlodawer and Sjolin (Wlodawer, A., and Sjolin, L. (1983) Biochemistry 22, 2720-2728) and has been refined at 1.8-A resolution to an agreement factor of 0.204. Most of the structure of the semisynthetic enzyme closely resembles that found in ribonuclease A with the synthetic peptide replacing the C-terminal elements of the naturally occurring enzyme. No redundant structure is seen; residues 114-118 of the larger chain and residues 111-113 of the peptide do not appear in our map. The positions of those residues at or near the active site are very similar to, if not identical with, those previously reported by others, except for histidine 119, which occupies predominantly the B position seen as a minor site by Borkakoti et al. (Borkakoti, N., Moss, D. S., and Palmer, R. A. (1982) Acta Crystallogr. Sect. B Struct. Crystallogr. Cryst. Chem. 38,2210-2217) and not at all by Wlodawer and Sjolin (1983).

Amino Acid Sequence↗
PubMed1985

A semisynthetic bovine pancreatic ribonuclease containing a unique nitrotyrosine residue.

A fully active semisynthetic ribonuclease, RNase 1-118:111-124, may be prepared by enzymatically removing six residues from the COOH terminus of the protein (positions 119-124) and then complementing the inactive RNase 1-118 with a chemically synthesized peptide containing the COOH-terminal 14 residues of the molecule (RNase 111-124) [M. C. Lin, B. Gutte, S. Moore, and R. B. Merrifield (1970) J. Biol. Chem. 245, 5169-5170]. Nitration of tyrosine-115 in the peptide followed by complex formation with RNase 1-118 affords a fully active enzyme containing a unique nitrotyrosine residue in a position which is known and which is very likely to be completely exterior to the active site region. The binding constant between the tetradecapeptide and RNase 1-118 (5 X 10(6) M-1 at pH 6.0) is not changed by the nitration. Crystals of the nitrated complex are isomorphous with those of RNase 1-118:111-124, for which a refined 1.8-A structure has recently been obtained.

Animals↗
PubMed1975

Multiple antigenic sites on an eicosapeptide. I. Precipitin studies in the goat.

Purified peptide 105-124, an antigenic determinant from the carboxy terminus ribonuclease, was found to form an immune precipitate with antibody to that region prepared by affinity chromatography from goat hyperimmune antiserum to reduced carboxymethylated ribonuclease (CM-RNase). Cm-rnase also gave an immune precipitate with the antibody. Purified antibody to another region of similar size (40-61) did not form a precipitate with CM-RNase but did co-precipitate in the presence of antibody to peptide 105-124 and CM-RNase. The precipitin reaction between antibody to peptide 105-124 and CM-RNase was inhibited by two synthetic derivatives, peptides 118-124 and ala114-RNase 114-124. Stoichiometry of the precipitin reactions of antibody to 105-124 with CM-RNase or peptide 105-124 suggested an antigen valency of three or more. Consistent with this both peptides 105-124 and ala114-RNase 114-124 elicited immediate cutaneous reactions but 118-124 did not. These findings suggest that the eicosapeptide 105-124 is multivalent since at least three antibodies can react simultaneously with it.

Animals↗