MECHANISMS OF STABILIZATION OF B-HEMEPROTEINS

B-血红素蛋白的稳定机制

基本信息

批准号：
6719095
负责人：
JULIETTE T LECOMTE
金额：
$ 21.68万
依托单位：
PENNSYLVANIA STATE UNIVERSITY, THE
依托单位国家：
美国
项目类别：
财政年份：
1990
资助国家：
美国
起止时间：
1990-07-01 至 2007-02-28
项目状态：
已结题

项目摘要

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：设计用于医学和其他领域的功能性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理化学性质。该提案旨在调查 b-血红蛋白微调能力所必需的因素识别并结合血红素基团。起始点是脱辅基蛋白细胞色素 b5，一种稍微稳定的球状蛋白，能够结合血红素基团可逆且具有高亲和力。血红素结合诱导结构变化，主要在结合位点的α螺旋中，并且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、光学光谱和多核核磁共振光谱来表征这些变化野生型脱辅基细胞色素 b5 及其变体。 NMR 弛豫和氢交换将用于探测空结合位点和折叠芯。将从变性中提取热力学参数实验和对假体基团的亲和力将通过研究血红素结合和释放的动力学。与全蛋白数据将提供结合和施加的扰动图血红素结合位点的全面能量描述设计。检验细胞色素的血红素结合环的假设作为一个自治模块，该循环将被插入到不同的蛋白质（具有 SH3 结构域拓扑的小亚基）。的属性将分析构建体并与原始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通过 FixL 的血红素结合位点的特征进行搜索，FixL 是一个根瘤菌氧传感器蛋白。这些人工收集的信息天然蛋白质将有助于定义b的构建原理适合设计人工血红素结合剂的血红素蛋白。