Structure Function Correlation of G-Proteins

warshelmcb - 0003872gtp结合蛋白在几乎所有生物体的细胞功能调控中发挥重要作用，并控制信号转导过程。获得这些蛋白质作用的详细分子图像是现代分子生物学的一个重要挑战。g蛋白的研究经历了一场“结构革命”，g蛋白及其与相应的辅助蛋白和/或过渡态类似物的复合物的新结构迅速出现。这为阐明这些蛋白质作用的详细分子机制提供了一个独特的机会。在过去的几年里，已经开发了将酶的结构与其功能联系起来的计算机模拟方法。这些方法在Ras催化反应的研究中是非常有用的。特别是，模拟表明GTP，而不是Gln61，是Ras酶促机制的实际基础。这被广泛认为是g蛋白和相关系统作用的可能机制。前人的研究还发现GTPase反应的速率常数与g-磷酸的pKa之间存在线性自由能关系（LFER）。该LFER为GTP作为基础方案提供了实验支持。先前的研究表明，GAP是通过结合两种效应来起作用的。首先，它主要使用Arg789对过渡态进行直接静电稳定。其次，它通过将Ras“推”到催化构型来间接起作用。这些模拟研究导致了当前的项目，该项目利用了g蛋白结构研究的巨大进展。在这个项目中，工作将在以下几个方向进行。(i)磷酸盐水解的潜在表面将得到验证和改进。计算和观测的LFER、同位素效应和激活熵将进行比较。水辅助过渡态的稳定性，包括相应的熵贡献也将被检查。这项工作将有助于解决机制问题，并在细化EVB表面为g蛋白的研究。（ii）研究GAP对Ras的激活作用，重点关注突变如Gln61和Gly12的影响。这些研究将检验这些残基是直接起作用还是通过稳定Ras的催化结构起作用。（iii）对不同g蛋白的作用进行比较研究，重点是转导蛋白、EF-Tu和Ras。这些蛋白质中GTPase反应的相对速率将被复制。这些研究将决定它们是否使用一种共同的机制。（iv）虽然g蛋白复合物和TS类似物提供的结构信息可能非常有用，但其解释并不简单，因为这些分子的电荷分布与实际TS的电荷分布之间的关系相当不清楚。因此，从头算和EVB计算将用于提取具有TS类似物的g蛋白的可用晶体结构所提供的信息。

WARSHELMCB-0003872GTP-binding proteins play an important role in the regulation of cellular functions in virtually all living organisms, and control signal transduction processes. Obtaining detailed molecular pictures of the actions of these proteins is an important challenge to modern molecular biology. The studies of G-proteins underwent a "structural revolution" where new structures of G-proteins and their complexes with the corresponding accessory proteins and/or transition state analogues are rapidly emerging. This offers a unique opportunity for elucidating the detailed molecular mechanisms of the action of these proteins.In the past years, computer simulation approaches that correlate the structures of enzymes with their functions have been developed. These methods were found to be very useful in the studies of the reaction catalyzed by Ras. In particular, the simulations suggested that the GTP, rather than Gln61, is the actual base in the enzymatic mechanism of Ras. This is now widely accepted as the likely mechanism for the action of G-proteins and related systems. The previous studies also led to the finding of a Linear Free Energy Relationship (LFER) between the rate constant of the GTPase reaction and the pKa of the g-phosphate. This LFER has given experimental support to the GTP as a base proposal. The previous studies demonstrated that the GAP operates by combining two effects. First, it acts by a direct electrostatic stabilization of the transition state using primarily Arg789. Second, it acts indirectly by "pushing" Ras to a catalytic configuration. These simulation studies lead to the current project that exploits the enormous progress in structural studies of G-proteins. In this project, the efforts will be in the following directions.(i) The potential surfaces for phosphate hydrolysis will be validated and refined. The calculated and observed LFER, isotope effects and activation entropies will be compared. The stability of water-assisted transition states, including the corresponding entropic contribution will also be examined. This effort will help in resolving mechanistic problems and in refining EVB surfaces for studies of G-proteins.(ii) The activation of Ras by GAP will be studied, with a focus on the effect of mutations such as Gln61 and Gly12. These studies will examine whether these residues act directly or by stabilizing the catalytic configuration of Ras.(iii) A comparative study of the action of different G-proteins will be conducted, focusing on transducin, EF-Tu, and Ras. The relative rates of the GTPase reaction in these proteins will be reproduced. These studies will determine whether or not they use a common mechanism.(iv) Although the structural information offered by complexes of G-proteins and TS analogues is potentially very useful, its interpretation is not straightforward because the relationship between the charge distribution of these molecules and that of the actual TS is rather unclear. Thus ab initio and EVB calculations will be used to extract the information offered by the available crystal structures of G-proteins with TS analogues.