MOLECULAR STUDIES OF TRP REPRESSOR STRUCTURE & FUNCTION

我们工作的长期目标是了解蛋白质结构 控制功能，以确定结构改变的蛋白质如何获得 新的属性。 这些变化可能是疾病状态的原因。 的 本提案的目的是检查结构、稳定性和折叠 机制在E. coliTrp阻遏物。 我们将使用分子遗传学， 生物化学来开发改变的阻遏物，并比较它们的稳定性， 折叠速率和结构与野生型相似。 这项工作将 围绕四个具体目标组织：三种类型的Trp诱变 R基因，以及改变Trp R蛋白的生化方法。 目标1. 一 温度敏感突变的选择将用于分离 在化学作用后携带热敏和冷敏阻遏物的突变体 trp R基因的诱变。 每种蛋白质对热的稳定性 和化学变性将使用圆二色性测定 通过分光偏振法（CD），并确定折叠速率。 结晶和核磁共振研究将用于确定结构 突变的后果。 这项工作的目标是获得一个 了解蛋白质的哪些氨基酸残基有助于 折叠状态的稳定性，以及它们是如何做到的。 目标2. 关键氨基酸 野生型色氨酸阻遏物疏水核心中的残基将被 从X射线晶体结构中鉴定出来。 最初，其中一个将 通过密码子随机化突变为所有19个其他残基， 抗坏血酸定向诱变。 阻遏物的表征 从初始突变体组中纯化的蛋白质将与所述的相似 上面的突变体。 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。 目标3. 特定的氨基酸置换将在预定的时间进行。 阻遏物的一个螺旋内的位点。 这个螺旋的动力学， 以及折叠速率和整体蛋白质稳定性，都受到 本网站的变化;对这些影响的解释将通过以下方式进行测试： 所描述的突变。 纯化的蛋白质的稳定性和折叠速率 突变蛋白将通过CD测量，其结构和动力学将 通过NMR进行分析。 这些实验测试了如何稳定的一个 单螺旋有助于蛋白质的整体稳定性， 研究这种稳定的详细机制。 目标4。 的 蛋白质将通过酶促蛋白水解和化学裂解来分解， 产生对应于或包含折叠单元的片段。 的 片段的稳定性和折叠速率以及重组时的稳定性和折叠速率， 通过CD测定，并通过NMR或X射线进行结构表征， 企图。 这些实验的目的是确定的定义， 以及蛋白质的折叠单元和结构域之间的关系，并且可以 为折叠途径的某些步骤提供了一个简单的模型。

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.