Investigation on Ligand Active Conformer of Bovine Myoglobin.

牛肌红蛋白羰基的一个单一配体结合位置已经显示出四个离散的快速相互转化构象(CI到CIV)(J.Biol.Chem)。257,11893)。这一发现是肌红蛋白分子必须是动态的，至少在配体结合部位的有力证据和一致，因为X射线衍射研究揭示的肌红蛋白结构不允许氧和一氧化碳等配体进入和离开分子。一个次要的构象IV(占蛋白质的2-3%)被认为是具有配基活性的，但其结构和反应活性仍有待研究。在本项目中，我们试图通过对肌红蛋白结构基因进行定点突变而使其成为肌红蛋白的主要构象，从而描述CIV的结构和反应性。根据Okayama-Berg的方法，从胎牛骨骼肌中提取Poly(A)^+-RNA，逆转录制备cDNA.…的杂交此外，用人肌红蛋白外显子II DNA片段制备的DNA片段显示牛肌红蛋白基因的大小约为1.1kb，这与Northern印迹分析确定的mRNA大小一致。将由核苷酸序列推导出的氨基酸序列与从纯化蛋白获得的氨基酸序列进行比较。差异在于：99个Val-&Gt；ILU，101个ILU-&Gt；Vol，122个Asn-&Gt；Asp，124个Ala-&Gt；Gly，129个Gly-&Gt；Ala，142个Ala-&Gt；Met，144个Glu-&Gt；Ala，145个Lys-&Gt；Gln。从牛心肌和携带肌红蛋白表达载体的酵母细胞中提纯的肌红蛋白在SDS-PAGE上显示了相同的N末端序列、脱氧形式的紫外可见光谱和相对分子质量。肌红蛋白基因在组(-&gt；Gln，Ser，Gly，Arg)和43Phe(-&gt；Ser)处发生突变。这些突变有望减弱或减少组氨酸()对CIV的不稳定作用。突变的肌红蛋白还没有得到纯化。我们正在继续开展这一项目。较少

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 … More 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