MECHANISMS OF STABILIZATION OF B-HEMEPROTEINS

B-血红素蛋白的稳定机制

• 批准号: 3464280
• 负责人: JULIETTE T LECOMTE
• 金额: $ 9.63万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-01 至 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3464280
• 关键词: binding proteins; chemical models; chemical stability; chimeric proteins; circular dichroism; conformation; cytochrome b; hemoprotein structure; laboratory rat; nuclear magnetic resonance spectroscopy; oxidation reduction reaction; protein reconstitution; thermodynamics

This proposal addresses two types of problems relevant to heme-containing proteins: general questions on the relationship between primary sequence and secondary and tertiary structure, and specific questions on the role of the prosthetic group in determining the structure and stability of the native material. The heme-containing hydrophilic domain of hepatic cytochrome b5 (cyt b5) is chosen as model system. Much is known about cyt b5 function and structure. Yet the nature and relative importance of the interactions between the prosthetic group and the protein matrix are not fully understood, and the structure dictated by the polypeptide chain alone has not been characterized in detail. Cyt b5 will be subjected to systematic studies aimed at defining the various interactions. (a)The apoprotein of cyt b5 will be investigated in detail by circular dichroic and proton nuclear magnetic resonance spectroscopy. The existing elements of secondary structure will be identified and the way in which they pack against each other described; the stability of the structure will be tested by varying external conditions; the kinetics of heme binding and its conformational consequences will be monitored. (b)Apocyt b5 will be reconstituted with protoporphyrin IX, i.e. the prosthetic group without the central iron atom. Structural data will be obtained on the "des-Fe" protein to extract the interactions between the protein and the protoporphyrin ring. (c)Single- and double-site mutant proteins will be produced and their apoprotein probed by the same methods as applied to the wild-type apoprotein. The mutants will reveal the contribution of selected amino acids to the structure of the apoprotein and its stability. The data collected in (a), (b), and (c) will be compared to the corresponding information available on the reduced and oxidized forms of the holoprotein. Thus the roles of amino acid sequence, prosthetic group, and iron atom in fixing the native holoprotein conformation and determining its thermodynamic properties will be characterized individually. The conclusions of this work will apply to a better understanding of the structural determinants of proteins.

该提案解决了与含血红素相关的两类问题 蛋白质：关于一级序列之间关系的一般问题 以及二级和三级结构，以及有关作用的具体问题 假体组决定了假体的结构和稳定性 原生材料。肝内含血红素的亲水结构域 选择细胞色素b5（cyt b5）作为模型系统。 关于 cyt b5 的功能和结构已知很多。然而大自然和 假肢组和假体组之间相互作用的相对重要性 蛋白质基质尚未完全了解，并且其结构由 单独的多肽链尚未被详细表征。细胞色素b5 将进行系统研究，旨在确定各种 互动。 (a)将通过循环详细研究细胞色素b5的脱辅基蛋白 二向色和质子核磁共振光谱。现有的 二级结构的元素将被识别，并以何种方式 所描述的它们相互竞争；结构的稳定性将 通过不同的外部条件进行测试；血红素结合动力学和 其构象后果将受到监测。 (b)Apocyt b5 将用原卟啉 IX 重建，即 没有中心铁原子的辅基。结构数据将是 在“des-Fe”蛋白质上获得，以提取之间的相互作用 蛋白质和原卟啉环。 (c)将产生单位点和双位点突变蛋白及其 采用与野生型相同的方法探测脱辅基蛋白 脱辅基蛋白。突变体将揭示所选氨基酸的贡献 酸对脱辅基蛋白的结构及其稳定性的影响。 (a)、(b) 和 (c) 中收集的数据将与 有关还原和氧化形式的相应信息 全蛋白。因此，氨基酸序列、辅基的作用， 和铁原子固定天然全蛋白构象并确定 其热力学性质将被单独表征。这 这项工作的结论将有助于更好地理解 蛋白质的结构决定因素。