| Budget Amount *help |
¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1987: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1986: ¥600,000 (Direct Cost: ¥600,000)
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| Research Abstract |
A single ligand binding site of bovine myoglobin carbonyl has been shown to exhibit four discrete rapidlly interconverting conformers (CI to CIV) (J.Biol.Chem. 257,11893). This finding is strong evidence for and consistent with that myoglobin molecule must be dynamic at least at the ligand binding site, since myoglobin structure revealed by X-ray diffraction studies does not allow ligand such as oxygen and carbon monoxide to enter and get out of the molecule. A minor conformer IV (2-3% of the protein) is considered to be ligand active, but its structure and reactivity are remained to study. In this project, we are trying to describe the structure and reactivity of CIV by making it a major conformer of myoglobin via amino acid substitutions accomplished as a result of site directed mutagenesis of the myoglobin structue gene.
cDNA was prepared according to the method of Okayama-Berg by reverse transcription of poly(A)^+-RNA isolated from fetal bovine skeltal muscle. Cross hybridization of
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the cDNA preparation with the human myoglobin ExonII DNA fragment revealed bovine myoglobin cDNA of <about>1.1 Kb which is consistent with the size of mRNA determined by Northern blot analysis. The amino acid sequence deduced from the nucleotide sequence was compared with that obtained from the purified protein. Differences found are:99 Val-> Ilu, 101 Ilu -> Vol, 122 Asn -> Asp, 124 Ala -> Gly, 129 Gly -> Ala, 142 Ala -> Met, 144 Glu -> Ala, 145 Lys -> Gln.
Myoglobin cDNA under the GAL 7 promoter was successfully expressed in yeast as an oxygenated form. Myoglobin purified from bovine heart muscle and yeast cells carrying the myoglobin expression vector exhibit the same N terminal sequence, UV-Vis-spectra of an exygenated form and molecular weight on SDS-PAGE. Myoglobin cDNA was mutated at 64 His ( -> Gln, Ser, Gly, Arg) and 43 Phe ( -> Ser). These mutations are expected to diminish or decrease a destabilizing effect of histidine (64) on CIV. Mutant myoglobins are not yet purified. We are continuing to carry out this project. Less
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