MECHANISMS OF STABILIZATION OF B-HEMEPROTEINS

B-血红素蛋白的稳定机制

• 批准号: 6519744
• 负责人: JULIETTE T LECOMTE
• 金额: $ 20.85万
• 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/6519744
• 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：为医疗和其他设计功能蛋白设计功能蛋白的能力 用途至关重要地了解氨基酸之间的关系 序列和物理化学特性。该提议旨在调查 B-食用蛋白对精细调整能力至关重要的因素 识别并结合血红素组。起点是来自 细胞色素B5，一种边缘稳定的球状蛋白，能够结合血红素基团 可逆性和高亲和力。血红素结合引起结构变化， 主要在绑定位点的α-螺旋中，并且整个整个都有动态变化 蛋白质。副投影将结合分子生物学，光学 光谱和多核NMR光谱法以表征这些变化 在野生型启示型色素B5和变体中。 NMR松弛和氢 交换将用于探测空绑定位点的运动和 折叠核心。热力学参数将从变性中提取 实验和对假体的亲和力将通过 血红素结合和释放动力学的研究。与 多蛋白蛋白数据将提供通过绑定和 在血红素结合网站中要利用的全面充满活力的描述 设计。为了测试细胞色素的血红素结合环的假设 作为自主模块的功能，该循环将被插入不同 蛋白质（具有SH3结构域拓扑的小亚基）。的特性 将分析构建体并将其与原始父蛋白进行比较。 有效识别和结合血红素的替代机制 将通过fixl的血红素结合位点的特征来搜索 根瘤菌氧传感器蛋白。这些人造的信息 天然蛋白质将有助于定义B的构建原理 血蛋白适用于人工血红素粘合剂的设计。