STRUCTURE-ENERGY CORRELATES IN ASPARATE TRANSCARBAMYLASE

这项研究计划的长期目标是了解 多亚单位蛋白质和其他大分子组合的调节。 我们目前的目标是阐明大肠杆菌的变构机制。 天冬氨酸氨基转移酶的功能能量学分析 蛋白质-蛋白质和蛋白质-配基相互作用及其相关性 能量和结构信息。对宏观经济运行的分析 完成了该体系的热力学性质和原子 三个主要构象状态的坐标现在是可用的，使得 有可能开始绘制能量转导的路径图 结构。下一个赠款期间的主要目标将是：(A)继续 低压和高压分析胶色谱技术的发展 方法，并将它们应用于以前无法获得的蛋白质的分析- 蛋白质在催化亚基(C3)、调节亚基(R2)中的相互作用 和天然酶(C6R6)在一系列条件下。(B)延长 静电效应的实验分析(I) 综合分析了不同的pH、离子强度和 特定离子对结构、结构动力学、能量学和 野生型ATCase及其亚基的功能：(Ii)验证假说 在前一批赠款期间由计算机模拟开发，通过检查 单位点突变体的结构、能量学和功能性质 其中被计算为在组装时经历较大PK变化的组或 修饰了配体结合，(Iii)研究了其作用机制。 CTP和UTP的协同抑制作用，特别是电离物质的作用 组，通过定点突变和酶促试验。(C)发展 冷冻等电聚焦法作为一种分析结扎细胞群体的方法 物种并分析一系列PALA连接物种的种群 PH和温度，以及CTP和ATP的存在和不存在时。(D) 建立变构机理的统计热力学模型。

The long range goal of this research program is to understand mechanisms of regulation in multisubunit proteins and other macromolecular assemblies. Our immediate goal is to elucidate the allosteric mechanism of E. coli aspartate transcarbamylase via analysis of the functional energetics of protein-protein and protein-ligand interactions and correlation of energetic and structural information. An analysis of the macroscopic thermodynamic properties of this system has been completed and atomic coordinates for three major conformational states are now available, making it possible to begin mapping pathways of energy transduction within the structure. Major goals of the next grant period will be to: (a) Continue the development of low and high pressure analytical gel chromatographic methods and apply them to the analysis of previously inaccessible protein- protein interactions in the catalytic subunit (c3), regulatory subunit (r2) and native enzyme (c6r6) over a range of conditions. (b) Extend the experimental analysis of electrostatic effects by (i) carrying out a comprehensive analysis of the effects of varying pH, ionic strength and specific ions on the structure, structural dynamics, energetics and function of wild type ATCase and its subunits, (ii)testing hypotheses developed by computer modelling in the previous grant period by examining the structure, energetics and functional properties of single site mutants in which groups calculated to undergo large pK changes upon assembly or ligand binding are modified, (iii) investigating the mechanism of synergistic inhibition by CTP and UTP, particularly the role of ionizable groups, by site-directed mutagenesis and enzymatic assay. (c) Develop cryoisoelectric focussing as a method for analyzing populations of ligated species and analyze the populations of PALA-ligated species over a range of pH and temperature and in the presence and absence of CTP and ATP. (d) Develop statistical thermodynamic models of the allosteric mechanism.