MOLECULAR STUDIES OF TRP REPRESSOR STRUCTURE & FUNCTION

TRP 阻遏物结构的分子研究

• 批准号: 3468005
• 负责人: Jannette Carey
• 金额: $ 12万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-01-01 至 1994-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3468005
• 关键词: DNA binding protein; X ray crystallography; chemical stability; circular dichroism; nuclear magnetic resonance spectroscopy; protein folding; protein reconstitution; protein sequence; protein structure function; proteolysis; reversed phase chromatography; temperature sensitive mutant; transcription factor

The long-range goal of our work is to understand how protein structure governs function, to determine how proteins with altered structures acquire new properties. Such changes can be responsible for disease states. The objective of this proposal is to examine structure, stability, and folding mechanism in E. coli trp repressor. We will use molecular genetics and biochemistry to develop altered repressors, and compare their stabilities, folding rates, and structures with those of wild-type. The work will be organized around four specific aims: three kinds of mutagenesis of the trp R gene, and a biochemical approach to alter the Trp R protein. Aim 1. A selection for temperature-sensitive mutations will be used to isolate mutants bearing heat- and cold-sensitive repressors following chemical mutagenesis of the trp R gene. The stability of each protein to thermal and chemical denaturation will be determined using circular dichroism spectropolarimetry (CD), and folding rates will be determined. Crystallization and NMR studies will be used to determine the structural consequences of mutation. The goal of this work is to gain an understanding of which amino acid residues of a protein contribute to the stability of the folded state, and how they do so. Aim 2. Key amino acid residues in the hydrophobic core of wild-type trp repressor will be identified from the x-ray crystal structure. Initially, one of these will be mutated to all 19 other residues by codon randomization using oligonucleotide-directed mutagenesis. Characterization of the repressors purified from an initial group of mutants will be similar to that described for ts mutants above. These experiments should extend our understanding of the nature of the hydrophobic core and its role in folding and stability. Aim 3. A specific amino acid replacement will be made at a predetermined site within one helix of the repressor. The dynamics of this helix, as well as folding rates and overall protein stability, are all affected by changes at this site; an explanation for these effects will be tested by the mutation described. The stability and folding rates of the purified mutant protein will be measured by CD, and its structure and dynamics will be analyzed by NMR. These experiments test how the stabilization of a single helix contributes to the overall stability of the protein, and examine the detailed mechanisms for that stabilization. Aim 4. The protein will be dissected by enzymatic proteolysis and chemical cleavage to generate fragments corresponding to or containing folding units. The stabilities and folding rates of fragmentsalone and when recombined will be determined by CD, and structural characterization by NMR or x-ray will be attempted. These experiments are aimed at determining the definitions of, and relationship between, folding units and domains of the protein, and may provide a simple model for certain steps of the folding pathway.

我们工作的远程目标是了解蛋白质结构如何 控制功能，以确定结构改变的蛋白质如何获得 新属性。 这种变化可能导致疾病状态。 这 该建议的目的是检查结构，稳定性和折叠 大肠杆菌TRP阻遏物中的机制。 我们将使用分子遗传学和 生物化学以开发改变的阻遏物，并比较其稳定性 折叠率，以及与野生型的结构。 工作将是 围绕四个特定目的组织：TRP的三种诱变 R基因和一种改变TRP R蛋白的生化方法。 目标1 选择温度敏感的突变将用于分离 化学后带有热敏阻遏物的突变体 Trp r基因的诱变。 每种蛋白质对热的稳定性 化学变性将使用圆形二色性确定 光谱极化（CD）和折叠速率将确定。 结晶和NMR研究将用于确定结构 突变的后果。 这项工作的目的是获得 了解蛋白质的哪种氨基酸残基有助于 折叠状态的稳定性，以及它们的稳定性。 目标2。钥匙氨基酸 野生型TRP阻遏物的疏水核心中的残留物将是 从X射线晶体结构中标识。 最初，其中之一 使用密码子随机分组将所有19个残基突变 寡核苷酸指导的诱变。 阻遏物的特征 从初始突变体纯化将与所描述的相似 对于上面的TS突变体。 这些实验应该扩展我们对 疏水核的性质及其在折叠和稳定性中的作用。 AIM 3。将在预先确定的 站点在阻遏物的一个螺旋中。 这个螺旋的动态， 以及折叠率和整体蛋白质稳定性都受到 在此网站上发生变化；这些效果的解释将由 描述的突变。 纯化的稳定性和折叠率 突变蛋白将通过CD测量，其结构和动力学将 通过NMR分析。 这些实验测试了A的稳定 单螺旋有助于蛋白质的整体稳定性，并且 检查该稳定的详细机制。 目标4 蛋白质将通过酶促蛋白水解和化学裂解来解剖蛋白质 生成对应于或包含折叠单元的片段。 这 碎片的稳定性和折叠率，重新组合时将是 由CD确定，NMR或X射线的结构表征将是 尝试。 这些实验旨在确定， 以及蛋白质的折叠单元和域之间的关系，可能 为折叠路径的某些步骤提供一个简单的模型。