MECHANISMS OF STABILIZATION OF B-HEMEPROTEINS

B-血红素蛋白的稳定机制

• 批准号: 6636187
• 负责人: JULIETTE T LECOMTE
• 金额: $ 21.69万
• 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 2005-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6636187
• 关键词: bacterial proteins; chemical models; chemical stability; cofactor; combinatorial chemistry; computer simulation; conformation; cytochrome b5 reductase; heme; hemoprotein; iron; molecular dynamics; molecular site; nuclear magnetic resonance spectroscopy; peptide chemical synthesis; protein binding; protein folding; protein structure function; proteomics; recombinant proteins; site directed mutagenesis; structural biology; thermodynamics

DESCRIPTION: The ability to design functional proteins for medical and other uses depends critically on understanding the relationship between amino acid sequence and physico-chemical properties. This proposal seeks to investigate the factors essential to the finely tuned capacity of b-hemoproteins to recognize and bind the heme group. The starting point is the apoprotein from cytochrome b5, a marginally stable globular protein able to bind the heme group reversibly and with high affinity. Heme binding induces structural changes, mostly in the alpha-helices of the binding site, and dynamic changes throughout the protein. The subprojects will combine molecular biology, optical spectroscopy, and multinuclear NMR spectroscopy to characterize these changes in wild-type apocytochrome b5 and variants. NMR relaxation and hydrogen exchange will be used to probe the motions of the empty binding site and the folded core. Thermodynamic parameters will be extracted from denaturation experiments and the affinity for the prosthetic group will be determined with a study of the kinetics of heme binding and release. Comparison with the holoprotein data will provide a map of the perturbations imposed by binding and a comprehensive energetic description to be exploited in heme-binding site design. To test the hypothesis that the heme binding loop of the cytochrome functions as an autonomous module, this loop will be inserted into a different protein (a small subunit with the topology of an SH3 domain). The properties of the constructs will be analyzed and compared to the original parent proteins. Alternative mechanisms for the efficient recognition and binding of the heme will be searched with the characterization of the heme-binding site of FixL, a rhizobial oxygen sensor protein. The information gathered on these artificial and natural proteins will help define the principles of construction of b hemoproteins suitable for the design of artificial heme-binders.

描述：设计用于医疗和其他领域的功能蛋白质的能力 用途关键取决于对氨基酸之间关系的理解 序列和物化性质。这项提议旨在调查 B-血球蛋白微调能力的关键因素 识别并绑定血红素组。起始点是来自 细胞色素b5，一种略微稳定的球状蛋白，能够结合血红素基团 可逆的，高亲和力的。血红素结合会引起结构变化， 主要是在结合部位的α-螺旋中，并在整个过程中动态变化 蛋白质。这些子项目将结合分子生物学、光学 光谱学和多核核磁共振波谱来表征这些变化 野生型凋亡素b5及其变异体。核磁共振弛豫与氢 交换将用于探测空结合部位的运动和 折叠芯。热力学参数将从变性过程中提取 实验和对假体基团的亲和力将通过 血红素结合与释放的动力学研究。与 全蛋白数据将提供由结合和 血红素结合部位有待开发的综合能量学描述 设计。来检验细胞色素的血红素结合环的假设 作为一个自主模块，此循环将被插入到不同的 蛋白质(具有SH3结构域拓扑结构的小亚基)。的属性 这些构建物将被分析，并与原始的亲本蛋白质进行比较。 有效识别和结合血红素的替代机制 将用FixL的血红素结合位点的特征进行搜索，a 根瘤菌氧感受器蛋白。在这些人造生物上收集的信息 天然蛋白质将有助于确定b的构建原理。 适合于设计人造血红素粘合剂的血红素蛋白。