MOLECULAR STUDIES OF PROTEIN STRUCTURE AND FUNCTION

该研究实验室工作的长期目标是 更好地理解蛋白质结构与 功能，包括氨基酸序列与 高级结构和生物化学与 生理功能 与疾病状态相关的蛋白质改变 通常显示新属性。 更好地了解新的 获得的功能可能会导致洞察他们如何最终 控制在 我们研究蛋白质结构的一般方法， 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，评估它们在体内和体内的功能。 通过生物化学和遗传分析体外，并确定其结构 在所需的分辨率通过各种生物物理方法。 在 目前的竞争性更新，我们建议继续我们正在进行的研究 的折叠，稳定性和动力学，在三个蛋白质系统，E。大肠杆菌TR 阻遏蛋白（TrpR）E.大肠杆菌精氨酸阻遏物（ArgR）和酵母iso-1- 细胞色素C（cytc）。 我们计划测试有序的自我组装 的TrpR蛋白水解片段是一个很好的模型折叠途径， 完整的TrpR，通过使用手动混合和/或停流CD，UV，和 荧光光谱比较片段组装的动力学 与完整TrpR的折叠动力学。 检查联轴器 之间的结构动力学，热稳定性，和功能的TrpR，我们 将对纯蛋白分离物进行量热和核磁共振实验 温度敏感型TrpR突变体 我们将测试一个 TrpR二聚体自缔合中结构重要残基 随机化其密码子，在体内确定每个突变体的表型 并通过交联、凝胶电泳和凝胶渗透色谱法检测纯化的突变蛋白， 过滤和荧光各向异性。 审查独立性 蛋白质折叠结构域 和稳定性，我们将比较 结构、稳定性、多聚体组装状态和折叠机构 嵌合TrpR衍生物与野生型TrpR衍生物的比较， 生物化学方法和光谱学。 我们将测试 ArgR结构域组织的初步模型，通过纯化N- 末端片段，并将其生化特性与 完整的ArgR。 我们计划制备同位素标记的ArgR片段， 尝试核磁共振结构测定域。 的序列和 第一步中螺旋配对反应的结构要求 的酵母iso-1-cytc折叠途径将进行检查， 定向突变，纯化和解剖蛋白质， 通过CD、UV、荧光， 量热法和NMR。

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.