MUTANTS OF REPRESSOR AND PERIPLASMIC BINDING PROTEINS

阻遏蛋白和周质结合蛋白的突变体

基本信息

批准号：
3287298
负责人：
KATHLEEN S MATTHEWS
金额：
$ 12.11万
依托单位：
RICE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1985
资助国家：
美国
起止时间：
1985-08-30 至 1990-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/3287298
关键词：
Escherichia coli bacterial genetics crystallization gene mutation genetic mapping ligands mutant

项目摘要

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.

周质结合蛋白中的独特结构特征和将使用该方法探索来自大肠杆菌的乳糖和TRP阻遏物定点诱变以生成具有特定改变的蛋白质在蛋白质的主要序列中所需的点。这些蛋白质在细菌细胞中起重要的非酶作用。详细的三维结构数据可用于阿拉伯糖结合蛋白质。基于与阿拉伯糖结合的主要序列同源性蛋白质，一种乳糖抑制蛋白的糖结合位点预测，以及与其他DNA结合位点具有同源性的区域已经发现了LAC和TRP阻遏物的阻遏物。突变体产生的蛋白质将大量隔离并表征关于其特性广泛的特性，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。选择诱变的位点将基于结合蛋白的3维结构和糖和DNA结合位点同源性与已知结构的蛋白质阻碍者。努力将针对绑定的变化所有蛋白质的位置，在两个域之间的铰链区域在结合蛋白中发现，在这些结合蛋白之间发现域。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；这预测的变化将与实验结果进行比较。突变蛋白的结晶（包括LAC抑制剂）将是试图将结构差异直接与父野型蛋白。结构和功能的结合来自此范围的不同蛋白质的数据将在确定特定氨基酸对折叠模式的影响和这些蛋白质结合的化学。