Biomedical subjects

T C Pochapsky

Publications and source records attributed to T C Pochapsky.

25 records · Page 2Linked to original sources

PubMed1993

Interpretation of hyperfine shift patterns in ferricytochromes b5 in terms of angular position of the heme: a sensitive probe for peripheral heme protein interactions.

The 1H-NMR hyperfine shift pattern of the heme in a variety of low-spin ferricytochromes b5 has been analyzed in terms of the angular position of the prosthetic group within a structurally and magnetically-conserved protein matrix. A simple model is presented in which the changes in the spread of the predominantly contact shifted methyl and predominantly dipolar shifted meso-H signals of the heme, as well as shift trends for individual signals, provide sensitive indicators of the orientation of the heme relative to the orbital hole (singly-occupied d orbital), which in turn is related to the rhombic magnetic axes. The invariance of the axial His and non-coordinated residue hyperfine shifts show that it is the heme within a relatively rigid protein matrix, rather than the magnetic coordinate system, which is displaced angularly about the heme normal in order to accommodate variations in the polypeptide, orientation of the heme about the alpha,gamma-meso axis, and the length of heme carboxylate chains. Native heme shows increased counterclockwise rotation about the heme normal in the order rat-->beef-->chicken ferricytochrome b5, which is attributed largely to increased bulk of a variable sequence hydrophobic cluster consisting of residues 23, 25 and 32. The two alternate heme orientations about the alpha,gamma-meso axis are shown to also differ by rotation about the heme normal. A semiquantitative estimate of the degree of angular accommodation based on the spread of the meso-H rhombic dipolar shifts indicate rotations of 2-10 degrees. Possible functional consequences of such angular accommodation in relation to the role of these proteins in electron transfer are discussed.

Animals↗

PubMed1992

1H NMR sequential assignments and identification of secondary structural elements in oxidized putidaredoxin, an electron-transfer protein from Pseudomonas.

Sequential 1H resonance assignments and secondary structural features of putidaredoxin (Pdx), a 106-residue globular protein consisting of a single polypeptide chain and a [2Fe-2S] cluster, are reported. No crystal structure has been obtained for Pdx or for any closely homologous protein. The sequentially assigned resonances represent ca. 83% of all the protons in Pdx and a large majority of those protons which are unaffected by the paramagnetism of the iron-sulfur cluster. A total of three alpha-helices, two beta-sheets, and two type I beta-turns have been identified from NOE (nuclear Overhauser effect) patterns. Besides the extensive beta-sheet described previously, a second sheet is identified, consisting of two short antiparallel strands (Ile 89-Thr 91 and Val 21-Leu 23), one of which ends in a tight type I turn (Thr 91-Pro 92-Glu 93-Leu 94). One short helix (Ala 26-Gly 31) and a second longer helical region (Glu 54-Cys 73) are present. This second helical region is discontinuous, breaking at Pro 61, resuming at Glu 65, and ending at Cys 73. The functionally important C-terminal tryptophan residue has been identified, and some structural constraints on this residue are described. Previously reported functional data concerning Pdx are discussed in light of present structural information. Finally, approaches to the determination of a high-resolution solution structure of the protein are discussed.

Amino Acid Sequence↗

PubMed1992

A chromatographic approach to the determination of relative free energies of interaction between hydrophobic and amphiphilic amino acid side chains.

A liquid chromatographic stationary phase was prepared by covalently binding to the surface of microparticulate silica gel functionality (benzylsilane), which mimics the side chain of the amino acid phenylalanine. The chromatographic retentions of the N-acetyl C-(N'-methyl) amides of various hydrophobic and amphiphilic amino acids on this stationary phase were measured using an aqueous mobile phase. A retention order of Gly < Ala < Cys < Val < Met < Pro < Ile < Leu < Tyr < Phe < Trp is seen at room temperature. Chromatographic retentions were used to derive free energies of adsorption of the amino acid derivatives on the chromatographic support relative to that of the glycine derivative. The temperature dependencies of the retention of aromatic and aliphatic amino acid derivatives differ in curvature, indicating a qualitative difference in the absorption mechanism. An adsorption model for retention is proposed, and arguments are made as to the suitability of an adsorption model for describing the contacts between amino acid side chains during the initial steps of protein folding.

Acetylation↗

PubMed1991

1H NMR identification of a beta-sheet structure and description of folding topology in putidaredoxin.

Putidaredoxin (Pdx), a 106-residue globular protein consisting of a single polypeptide chain and a [2Fe-2S] cluster, is the physiological reductant of P-450cam, which in turn catalyzes the monohydroxylation of camphor by molecular oxygen. No crystal structure has been obtained for Pdx or for any closely homologous protein. The application of two-dimensional 1H NMR methods to the problem of structure determination in Pdx is reported. A beta-sheet consisting of five short strands and one beta-turn has been identified from distinctive nuclear Overhauser effect patterns. All of the backbone resonances and a majority of the side-chain resonances corresponding to protons in the beta-sheet have been assigned sequence specifically. The sheet contains one parallel and three antiparallel strand orientations. Hydrophobic side chains in the beta-sheet face primarily toward the protein interior, except for a group of three valine side chains that are apparently solvent exposed. The potential significance of this "hydrophobic patch" in terms of biological activity is discussed. The folding topology, as determined by the constraints of the beta-sheet, is compared with that of other [2Fe-2S] proteins for which folding topologies are known.

Amino Acid Sequence↗

PubMed1990

1H NMR study of the influence of hydrophobic contacts on protein-prosthetic group recognition in bovine and rat ferricytochrome b5.

The proton nuclear magnetic resonance spectra of the soluble fragment of native bovine and genetically engineered wild-type rat ferricytochrome b5 reconstituted with a wide variety of hemes chemically modified at 2- and/or 4-positions have been recorded and analyzed. While all but one nonsymmetric heme yielded comparable amounts of the two heme orientations immediately after reconstitution, the relative proportion of the two orientations at equilibrium varied widely. The unpaired spin density distribution in the heme pi system leads to substituent hyperfine shift patterns in these paramagnetic complexes that are completely diagnostic of the heme orientation in the protein matrix. An empirical assignment strategy is outlined and applied which allows unequivocal assignment of the absolute orientation of a derivatized heme within the protein matrix. Using a series of hemes lacking 2-fold symmetry solely due to a single substitution, the preferences for localized site occupation of vinyls, methyls, and hydrogens are developed. The large differences in relative stability of the two orientations of native protohemin in the two cytochromes b5 is shown to result from the additivity of localized effects for the bovine protein and the near cancellation of competing effects in the rat protein. The major determinant of the heme orientation is judged to be a repulsive interaction between a vinyl and a hydrophobic cluster of amino acids including positions 23 and 25. The differences in this heme orientational preference among bovine, rat, and chicken ferricytochromes b5 could be correlated with the relative steric bulk of the residues at positions 23 and 25.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

Probing the mechanisms of macromolecular recognition: the cytochrome b5-cytochrome c complex.

The specificity of complex formation between cytochrome b5 (cyt b5) and cytochrome c (cyt c) is believed to involve the formation of salt linkages between specific carboxylic acid residues of cyt b5 with lysine residues on cyt c. Site-directed mutagenesis was used to alter the specified acidic residues of cyt b5 to the corresponding amide analogues, which resulted in a lower affinity for complex formation with cyt c. The dissociation of the complex under high pressure resulted in specific volume changes, the magnitude of which reflected the degree of solvation of the acidic residues in the proposed protein-protein interface.

Animals↗

PubMed1987

Separation of some enantiomeric di- and tripeptides on chiral stationary phases.

The chromatographic behavior of the N-3,5-dinitrobenzoyl derivatives of twelve dipeptide esters and two tripeptide esters was investigated on three different chiral stationary phases (CSPs). It is observed that the stereoisomers present in each sample may be cleanly separated on each chiral phase. A degree of regularity is noted in the elution order of the enantiomers and often of the diastereomers. Elution order of the enantiomers is related to a chiral recognition model for each CSP.

Amino Acids↗