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.

独特的结构特征在质周结合蛋白和