MUTANTS OF REPRESSOR AND PERIPLASMIC BINDING PROTEINS

阻遏蛋白和周质结合蛋白的突变体

• 批准号: 3287296
• 负责人: KATHLEEN S MATTHEWS
• 金额: $ 14.61万
• 依托单位: RICE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-08-30 至 1988-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3287296
• 关键词: Escherichia coli; bacterial genetics; crystallization; gene mutation; genetic mapping; ligands; mutant

Unique structural features in the periplasmic binding proteins and in the lactose and trp repressors from E. coli will be explored using the method of site-directed mutagenesis to generate proteins with specific alterations at desired points in the primary sequence of the protein. These proteins play significant non-enzymatic roles in the bacterial cell. Detailed three-dimensional structural data are available for the arabinose binding protein. Based on primary sequence homology with arabinose binding protein, a sugar binding site for the lactose represssor protein has been predicted, and a region with homology to DNA binding sites in other represssors has been found for both lac and trp repressors. The mutant proteins produced will be isolated in large quantities and characterized extensively with regard to their properties, including both equilibrium and kinetic measurements of binding, spectroscopic analysis, and chemical reactivity of selected amino acids. Selection of sites for mutagenesis will be based on the 3-dimensional structure of the binding protein and on sugar and DNA binding site homology with proteins of known structure for the repressors. Efforts will be directed toward changes in the binding sites of all the proteins, in the hinge region between the two domains found in the binding protein, and in the contact areas between these domains. The specific amino acid changes generated will be based on anticipated interesting alterations in the protein structure/function; the predicted changes will be compared to the experimental results. Crystallization of the mutant proteins (including lac repressor) will be attempted in order to directly compare structural differences with the parent wild-type protein. The combination of structural and functional data from this range of different proteins will be useful in determining the effects of specific amino acid changes on the folding patterns and chemistry of binding for these proteins.

周质结合蛋白的独特结构特征 将使用该方法探索来自大肠杆菌的乳糖和色氨酸阻遏物。 定点突变以产生具有特定改变的蛋白质 在蛋白质的初级序列中的所需位置。这些蛋白质 在细菌细胞中扮演着重要的非酶作用。详细 阿拉伯糖结合的三维结构数据是可用的 蛋白。基于与阿拉伯糖结合的一级序列同源性 蛋白，乳糖抑制蛋白的一个糖结合部位已经被 以及与其他DNA结合位点具有同源性的区域 已经发现了乳胶和色氨酸抑制物的抑制物。变种人 生产的蛋白质将被大量分离和鉴定 广泛地关于它们的性质，包括平衡和 结合、光谱分析和化学的动力学测量 选定氨基酸的反应性。诱变地点的选择 将基于结合蛋白的三维结构和 糖和DNA结合位点与已知结构的蛋白质同源性 压抑者。努力的方向将是改变约束性 所有蛋白质的位置，在两个结构域之间的铰链区 发现在结合蛋白中，以及它们之间的接触区域 域名。产生的特定氨基酸变化将基于 预期的蛋白质结构/功能的有趣变化； 预测的变化将与实验结果进行比较。 突变蛋白(包括lac抑制物)的结晶将是 尝试将结构差异与 亲本野生型蛋白。结构与功能的结合 来自这一系列不同蛋白质的数据将有助于确定 特定氨基酸的变化对折叠模式和 与这些蛋白质结合的化学。