MUTANTS OF REPRESSOR AND PERIPLASMIC BINDING PROTEINS

在周质结合蛋白和细胞外基质中的独特结构特征 乳糖和色氨酸阻遏物。大肠杆菌将使用该方法进行探索 定点突变产生具有特定改变的蛋白质 在蛋白质的一级序列中的所需点上。 这些蛋白质 在细菌细胞中发挥重要的非酶作用。 详细 三维结构数据可用于阿拉伯糖结合 蛋白 基于与阿拉伯糖结合的一级序列同源性 蛋白，乳糖阻遏蛋白的糖结合位点已经被 预测，和一个区域与DNA结合位点的同源性，在其他 已经发现了Lac和Trp阻遏物的阻遏物。 突变 产生的蛋白质将被大量分离并表征 广泛地关于他们的属性，包括平衡和 结合动力学测量、光谱分析和化学分析 选择的氨基酸的反应性。 诱变位点的选择 将基于结合蛋白的三维结构， 糖和DNA结合位点与已知结构的蛋白质同源， 抑制因子 将努力改变约束力 所有蛋白质的位点，在两个结构域之间的铰链区 在结合蛋白中，以及在这些蛋白之间的接触区域中， 域. 产生的特定氨基酸变化将基于 蛋白质结构/功能的预期有趣的变化; 将预测的变化与实验结果进行比较。 突变蛋白质（包括lac阻遏物）的结晶将是 试图直接比较结构差异与 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 特定氨基酸变化对折叠模式的影响， 结合这些蛋白质的化学过程。

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.