MECHANISMS OF STABILIZATION OF B-HEMEPROTEINS

描述：设计用于医疗和其他用途的功能蛋白质的能力 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 对b-血红素蛋白精细调节的能力至关重要的因素， 识别并结合血红素基团。起始点是来自 细胞色素b5，一种能够结合血红素基团的边缘稳定的球状蛋白质 可逆且具有高亲和力。血红素结合诱导结构变化， 主要是在结合位点的α-螺旋中， 蛋白质。这些子项目将结合联合收割机分子生物学、光学 光谱和多核NMR光谱来表征这些变化 在野生型脱辅基细胞色素B5和变体中。NMR弛豫和氢 交换将被用来探测空结合位点的运动， 折叠芯。从变性过程中提取热力学参数 实验和辅基的亲和力将用 血红素结合和释放的动力学研究。比较 全蛋白数据将提供结合所施加的扰动图， 一个全面的能量描述，以利用血红素结合位点 设计为了验证细胞色素的血红素结合环 作为一个自治模块，这个循环将被插入到一个不同的 蛋白（具有SH 3结构域拓扑结构的小亚基）。的性质 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 将搜索与血红素结合位点的FixL，一个 根瘤菌氧传感蛋白收集到的关于这些人工 天然蛋白质将有助于确定B的构建原则 适合于设计人工血红素结合剂的血红素蛋白。

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.