FOLDING AND STABILITY OF THE GENE V PROTEIN IN PHAGE F1

这项工作的目标是衡量对 通过特定的相互作用使蛋白质结构稳定 这些结构。这方面的知识将在 设计具有重要医学和工业价值的合成激素， 疫苗、调节蛋白和酶。 该项目将使用噬菌体的基因V(DNA结合蛋白) F1作为衡量氨基酸取代效应的模型系统 蛋白质的稳定性和结构。首先，我们计划获得一个 X-射线衍射法分析基因V蛋白的准确结构。 一个准确的模型对于解释我们以前的 实验和作为确定结构的起始模型 突变基因V蛋白。新的阶段性信息将从 几种技术，包括收集来自 含硒蛋氨酸基因V蛋白晶体的几个 波长。从这些程序中获得的模型将被改进 相对于1.8 angstroms的原始数据。 接下来，我们计划使用基因V蛋白的这种新结构来 解释我们之前的结果。我们建议利用这一结构来 提出一些假设，解释为什么一些基因V温度敏感突变体 已经改变了稳定性，以解释非极点到非极点的影响 掩埋地点的替换，并解释valine对 苏氨酸在内部位置的取代。 第三，我们将确定强稳定的晶体结构。 和不稳定的变种人。我们之前使用了一种遗传方法来 确定基因V中特别强的相互作用 蛋白。我们提议将我们已经提纯的11个突变体结晶 表现出与野生型非常不同的稳定性。使用 突变蛋白的结构，我们预计能够提示 其稳定性变化的结构性基础。 最后，我们将构建突变体来测量由于 基因V蛋白中的特定相互作用。我们建议衡量 由特定的相互作用对稳定性变化的贡献 构建其他突变体，这些突变体在属性中各不相同 例如静电相互作用或氢键，以及 确定它们的稳定性和结构，以及通过检查 控制在目标属性上没有不同的突变体。通过 比较特定交互在不同环境中的贡献 蛋白质中的上下文、几何或溶剂暴露 可以评估进行交互的要求。

The goal of this work is to measure the energetic contributions to the stabilization of protein structures made by specific interactions within these structures. This knowledge will be of considerable utility in the design of medically and industrially important synthetic hormones, vaccines, regulatory protein, and enzymes. This project will use the gene V (DNA-binding) protein of bacteriophage f1 as a model system to measure the effects of amino-acid substitutions on protein stability and structure. First, we plan to obtain an accurate structure of the gene V protein by x-ray diffraction analysis. An accurate model will be essential for interpretation of our previous experiments and as a starting model for determining the structures of mutant gene V proteins. New phasing information will be obtained from several techniques including collection of x-ray diffraction data from selenomethionine-containing gene V protein crystals at several wavelengths. A model obtained from these procedures will be refined against 1.8angstroms native data. Next we plan to use this new structure of the gene V protein to interpret our previous results. We propose to use this structure to formulate hypotheses as to why some gene V temperature-sensitive mutants have altered stabilities, to interpret effects of apolar-to-apolar substitutions at buried sites, and to interpret effects of valine to threonine substitutions at interior sites. Third, we will determine the crystal structures of strongly stabilized and destabilized mutants. We previously used a genetic approach to identify interactions that are particularly strong in the gene V protein. We propose to crystallize 11 mutants we have already purified that exhibit very different stabilities than the wild-type. Using the structures of the mutant proteins, we anticipate being able to suggest the structural basis of their changes in stability. Finally, we will construct mutants to measure energetic effects due to specific interations in the gene V protein. We propose to measure the contribution to changes in stability from specific interactions by constructing additional mutants which differ from each in the property of interest, such as electrostatic interactions or hydrogen bonding, and determining their stabilities and structures, as well as by examining control mutants that do not differ in the property of interest. By comparing the contributions of a particular interaction in different contexts within the protein, the geometric or solvent exposure requirements for making the interaction can be evaluated.