Investigation on Ligand Active Conformer of Bovine Myoglobin.

牛肌红蛋白配体活性构象的研究。

• 批准号: 61580238
• 负责人: SHIMADA Hideo
• 金额: $ 0.9万
• 依托单位: Keio University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1986
• 资助国家: 日本
• 起止时间: 1986 至 1987
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-61580238/
• 关键词: Bovine; Myoglobin; cDNA; Conformer; Expression; Yeast; Oxygenated Myoglobin; Nucleotide Sequence

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

牛肌红蛋白羰基的一个配体结合位点显示出四个离散的快速互转换构象（CI到CIV） （J.Biol.Chem.）。257年,11893年)。这一发现有力地证明了肌红蛋白分子至少在配体结合位点是动态的，因为x射线衍射研究揭示的肌红蛋白结构不允许配体（如氧气和一氧化碳）进入和离开分子。次要的构象IV（2-3%的蛋白质）被认为是配体活性的，但其结构和反应性仍有待研究。在这个项目中，我们试图描述CIV的结构和反应性，通过位点定向诱变肌红蛋白结构基因的氨基酸取代，使其成为肌红蛋白的主要构象。从胎牛骨骼肌中分离poly(A)^+-RNA，采用Okayama-Berg法逆转录制备cDNA。与人肌红蛋白ExonII DNA片段进行交叉杂交，结果显示牛肌红蛋白cDNA长度< bb0 1.1 Kb，与Northern blot测定的mRNA大小一致。从核苷酸序列中推导出的氨基酸序列与纯化蛋白的氨基酸序列进行了比较。发现的差异有：99个Val-> Ilu， 101个Ilu -> Vol， 122个Asn -> Asp， 124个Ala -> Gly， 129个Gly -> Ala， 142个Ala -> Met， 144个Glu -> Ala， 145个Lys -> Gln。在gal7启动子下，肌红蛋白cDNA成功地在酵母中以氧合形式表达。从牛心肌中纯化的肌红蛋白和携带该表达载体的酵母细胞表现出相同的N端序列、氧合形式的紫外-可见光谱和SDS-PAGE分子量。肌红蛋白cDNA突变位点为64个His （-> Gln, Ser, Gly, Arg）和43个Phe （-> Ser）。这些突变有望减少组氨酸对CIV的不稳定作用（64）。突变的肌红蛋白尚未纯化。我们正在继续执行这个项目。少