STRUCTURE-ENERGY CORRELATES IN ASPARATE TRANSCARBAMYLASE

这项研究计划的长期目标是了解 调节多亚基蛋白质和其他大分子组装。 我们的近期目标是阐明E.杆菌 天冬氨酸转氨甲酰酶的功能能量学分析 蛋白质-蛋白质和蛋白质-配体相互作用及相关性 能量和结构信息。 宏观分析 该体系的热力学性质已经完成，原子 三种主要构象状态的坐标现在可用， 它可以开始映射内的能量转导途径 结构 下一个赠款期的主要目标是： 低、高压分析凝胶色谱的发展 方法，并将其应用于分析以前无法获得的蛋白质- 催化亚基（c3）、调节亚基（r2）中的蛋白质相互作用 和天然酶（C6R6）。(b)延长 静电效应的实验分析，通过（i）进行 全面分析了不同pH值、离子强度和 特定离子的结构，结构动力学，能量学和 野生型ATCase及其亚基的功能，（ii）检验假设 在上一个赠款期内通过计算机建模开发的， 单位点突变体的结构、能量学和功能特性 其中基团经计算在组装时经历大的pK变化，或 配体结合的修改，（iii）调查的机制， CTP和UTP的协同抑制作用，特别是可电离的 组，通过定点诱变和酶法测定。 (c)发展 冷冻等电聚焦作为分析连接的 物种，并分析一系列PALA连接物种的种群， pH和温度以及在CTP和ATP存在和不存在下。 （d）其他事项 发展别构机制的统计热力学模型。

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.