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D Shih

Publications and source records attributed to D Shih.

24 records · Page 2Linked to original sources
PubMed1990

Effect of the distal residues on the vibrational modes of the Fe-CO bond in hemoglobin studied by protein engineering.

Using an Escherichia coli gene expression system, we have engineered human hemoglobin (Hb) mutants having the distal histidine (E7) and valine (E11) residues replaced by other amino acids. The interaction between the mutated distal residues and bound carbon monoxide has been studied by Soret-excited resonance Raman spectroscopy. The replacement of Val-E11 by Ala, Leu, Ile, and Met has no effect on the v(C-O), v(Fe-CO) stretching or delta(Fe-C-O) bending frequencies in both the alpha and beta subunits of Hb, although some of these mutations affect the CO affinity as much as 40-fold. The strain imposed on the protein by the binding of CO is not localized in the Fe-CO bond and is probably distributed among many bonds in the globin. The replacement of His-E7 by Val or Gly brings the stretching frequencies v(Fe-CO) and v(C-O) close to those of free heme complexes. In contrast, the substitution of His-E7 by Gln, which is flexible and polar, produces no effects on the resonance Raman spectrum of either alpha- or beta-globin. The replacement of His-E7 of beta-globin by Phe shows the same effect as replacement by Gly or Val. Therefore, the steric bulk of the distal residues is not the primary determinant of the Fe-CO ligand vibrational frequencies. The ability of both histidine and glutamine to alter the v(C-O), v(Fe-CO), or delta(Fe-C-O) frequencies may be attributed to the polar nature of their side chains which can interact with bound CO in a similar manner.

Binding Sites↗
PubMed1989

NMR study of human mutant hemoglobins synthesized in Escherichia coli. Consequences of tyrosine alpha 42 substitutions.

The hydroxyl group of Tyr alpha 42 in human hemoglobin forms a hydrogen bond with the carboxylate of Asp beta 99 which is considered to be one of the most important hydrogen bonds for stabilizing the "T-state." However, no spontaneous mutation at position 42 of the alpha subunit has been reported, and the role of the tyrosine has not been tested experimentally. Two artificial human mutant hemoglobins in which Tyr alpha 42 was replaced by phenylalanine or histidine were synthesized in Escherichia coli, and their proton NMR spectra were studied with particular attention to the hyperfine-shifted and hydrogen-bonded proton resonances. The site-directed mutagenesis of the Tyr alpha 42----Phe removes the hydrogen bond described above and prevents transition to the T-state so that the mutant Hb is rather similar to the "R-state" even when deoxygenated. On the other hand, the mutation from tyrosine to histidine causes less drastic structural changes, and its quaternary and tertiary structures are almost the same as native deoxy-Hb A. This may be attributed to the formation of a new hydrogen bond between His alpha 1(42) and Asp beta 2(99). These observations indicate that the hydrogen bond formed between Tyr alpha 42 and Asp beta 99 is required to convert unliganded Hb to the T-state.

Escherichia coli↗
PubMed1988

Hemoglobin Brockton [beta 138 (H16) Ala----Pro]: an unstable variant near the C-terminus of the beta-subunits with normal oxygen-binding properties.

Hemoglobin Brockton [beta 138 (H16) Ala----Pro] is an unstable variant associated with a mild anemia. It has the same electrophoretic mobility as and cannot be resolved from Hb A. Oxygen affinity measurements of blood and hemolysate do not indicate biphasic oxygen saturation, showing that the functional properties of the variant are very similar to those of Hb A. This implies that the introduction of proline into the H-helix at position 138 does not disrupt the critical inter- and intrasubunit hydrogen bonds and salt bridges at the beta carboxyl-terminal dipeptide, since these polar interactions are essential for the normal oxygen-binding properties of hemoglobin. X-ray crystallographic data are consistent with these findings and show that the consequences of the beta 138 Ala----Pro substitution are almost entirely confined to the immediate vicinity of the mutation site. Instability probably results from the inability of a buried hydrogen bond to form between Pro 138 beta and Val 134 beta.

Amino Acid Sequence↗
PubMedDate not supplied

The role of high-density lipoproteins in oxidation and inflammation.

High-density lipoproteins (HDL) in the basal state are anti-inflammatory, capable of destroying oxidized lipids that generate an inflammatory response. However, HDL during acute inflammation are altered and become pro-inflammatory. This "chameleon-like" nature of HDL is considered to be due to the complex composition of HDL. The data reviewed here demonstrate the key role of HDL in modulating inflammation and its implications for atherogenesis.

Animals↗
PubMedDate not supplied

Distal residues in the oxygen binding site of haemoglobin studied by protein engineering.

The geometries of the Fe-O2 and Fe-CO bonds in myoglobin and haemoglobin differ significantly from those in free porphyrin model compounds. It has been suggested that steric hindrance by Val-E11 and His-E7 and a hydrogen bond between His-E7 and oxygen affect the geometry and electronic state of the Fe-ligand bond, and that these interactions may be important in controlling oxygen affinity. We have produced mutant haemoglobins in E. coli having Val(67 beta)E11 replaced by Ala, Met, Leu or Ile and His(58 beta)E7 by Gln, Val or Gly. We have studied the effect of these mutations on the equilibrium and kinetics of ligand binding. The conformation of the new side chains and their effect on the protein structure have been examined by X-ray crystallography, and the vibrational properties of the Fe-CO bond observed by resonance Raman spectroscopy. We found that the steric hindrance of ligand binding by the E11 residue and the polarity of the E7 residue in the beta subunit are critical for fine-tuning ligand affinity.

Escherichia coli↗