MOLECULAR STUDIES OF PROTEIN STRUCTURE AND FUNCTION

这个研究实验室工作的长期目标是 更好地理解蛋白质结构和蛋白质之间的关系 功能，包括氨基酸序列与 高阶结构及其与生物化学的关系 生理功能。与疾病状态相关的改变蛋白 通常会显示新属性。更好地了解新事物 获得的功能可能会导致洞察它们最终可能 被控制住。我们研究蛋白质结构和结构的一般方法 功能是利用分子遗传学和/或生物化学来获得 蛋白质或蛋白质片段，评估它们在体内和体内的功能 通过生化和遗传分析，确定它们的结构 通过各种生物物理方法达到所需的分辨率。在 目前正在进行竞争更新，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，E.ColiTr 抑制子(Trpr)、大肠杆菌Arg抑制子(ArgR)和酵母iso-1- 细胞色素c(Cytc)。我们计划测试有序的自组装是否 Trpr蛋白水解性片段的折叠路径是一个很好的模型 完整的TrPR，通过使用手动混合和/或停流CD、UV和 用荧光光谱仪比较片段组装的动力学 与完整Trpr的折叠动力学一致。要检查耦合，请执行以下操作 在结构动力学、热稳定性和trpr功能之间，我们 将对纯分离蛋白进行量热和核磁共振实验 来自一个温度敏感型Trpr突变体。我们将测试一个 Trpr二聚体自缔合中重要的结构残基 将其密码子随机化，确定每个突变体在体内的表型 并对纯化的突变体蛋白进行交联、凝胶等检测 过滤和荧光各向异性。考察…的独立性 蛋白质结构域在折叠和稳定性方面，我们将比较 结构、稳定性、多聚体组装状态和折叠机构 将嵌合Trpr衍生物与野生型Trpr的嵌合Trpr衍生物通过使用 生化方法和光学光谱分析。我们将测试一个 精制N-ArgR结构域组织的初步模型 末端片段及其生化性质的比较 完整的ArgR。我们计划制备同位素标记的ArgR片段，并 尝试对结构域进行核磁共振结构测定。该序列和 第一步螺旋配对反应的结构要求 酵母菌的iso-1-cytc折叠途径将通过制作位点- 定向突变，纯化和解剖蛋白质，并分析 CD、UV、荧光光谱检测其片段结合反应， 量热法和核磁共振。

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.