MOLECULAR STUDIES OF PROTEIN STRUCTURE AND FUNCTION

蛋白质结构和功能的分子研究

基本信息

批准号：
2634678
负责人：
Jannette Carey
金额：
$ 14.33万
依托单位：
PRINCETON UNIVERSITY
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：
1990-01-01 至 2000-12-31
项目状态：
已结题

项目摘要

The long-term objective of the work in this research laboratory is to better understand the relationship between protein structure and function, including as well the relationship of amino acid sequence to higher-order structure and the relationship between biochemical and physiological function. Altered proteins associated with disease states typically display new properties. A better understanding of how new functions are acquired may lead to insight into how they may eventually be controlled. Our general approach to studying protein structure and function is to use molecular genetics and/or biochemistry to obtain proteins or protein fragments, assess their functions in vivo and in vitro by biochemical and genetic assays, and determine their structures at the required resolution by various biophysical methods. In the present competitive renewal, we propose to continue our ongoing studies of folding, stability, and dynamics in three protein systems, E. coli tr repressor (TrpR) E. coli arg repressor (ArgR), and yeast iso-1- cytochrome c(cytc). We plan to test whether the ordered self-assembly of TrpR proteolytic fragments is a good model for the folding pathway of intact TrpR, by using manual-mixing and/or stopped-flow CD, UV, and fluorescence spectroscopies to compare the kinetics of fragment assembly with the folding kinetics of intact TrpR. To examine the coupling between structural dynamics, thermal stability, and function of TrpR, we will conduct calorimetry and NMR experiments on the pure protein isolate from a temperature-sensitive TrpR mutant. We will test the role of a structurally important residue in TrpR dimer self-association by randomizing its codon, determining the phenotypes of each mutant in vivo and examining the purified mutant proteins by crosslinking, gel filtration, and fluorescence anisotropy. To examine the independence of protein domains in folding and stability, we will compare the structure, stability, multimeric assembly state, and folding mechanism of a chimeric TrpR derivative with those of wildtype TrpR by using biochemical methods and optical spectroscopies. We will test a preliminary model for domain organization of ArgR by purifying an N- terminal fragment and comparing its biochemical properties with those of intact ArgR. We plan to prepare isotopically-labelled ArgR fragment and attempt an NMR structure determination of the domain. The sequence and structural requirements for a helix-pairing reaction in the first step of the yeast iso-1-cytc folding pathway will be examined by making site- directed mutations, purifying and dissecting the proteins, and analyzing their fragment association reactions by CD, UV, fluorescence, calorimetry, and NMR.

这项研究实验室工作的长期目标是更好地了解蛋白质结构与功能，包括氨基酸序列与高阶结构以及生化与生理功能。与疾病状态相关的蛋白质改变通常显示新属性。更好地理解如何新获取功能可能会导致深入了解它们最终如何被控制。我们研究蛋白质结构和功能是使用分子遗传学和/或生物化学来获得蛋白质或蛋白质片段，评估其在体内的功能通过生化和遗传测定的体外，并确定其结构通过各种生物物理方法所需的分辨率。在目前的竞争性更新，我们建议继续我们正在进行的研究三种蛋白质系统中的折叠，稳定性和动力学抑制剂（TRPR）大肠杆菌ARG Repressor（ARGR）和酵母ISO-1- 细胞色素C（CYTC）。我们计划测试有序的自组装 TRPR蛋白水解片段是折叠途径的好模型完整的TRPR，使用手动混合和/或停止流量CD，UV和荧光光谱法比较碎片组装的动力学具有完整trpr的折叠动力学。检查耦合在结构动力学，热稳定性和TRPR的功能之间，我们将在纯蛋白分离株上进行量热法和NMR实验来自温度敏感的TRPR突变体。我们将测试一个通过结构上重要的残留物在TRPR二聚体自我关联中随机对其密码子进行随机，确定体内每个突变体的表型并通过交联，凝胶检查纯化的突变蛋白过滤和荧光各向异性。检查独立性折叠和稳定性中的蛋白质结构域，我们将比较结构，稳定性，多聚体装配状态和折叠机制通过使用WildType TRPR的嵌合TRPR衍生物生化方法和光谱法。我们将测试通过净化n-，用于ARGR领域组织的初步模型末端碎片并将其生化特性与完整的argr。我们计划准备同位素标记的ARGR片段，尝试对域的NMR结构确定。序列和第一步的螺旋配对反应的结构要求酵母ISO-1-CYTC折叠途径将通过使位点进行检查定向突变，净化和解剖蛋白质并分析它们通过CD，UV，荧光，碎片关联反应量热法和NMR。