Structure Function Correlation of G-Proteins

G 蛋白的结构功能相关性

• 批准号: 0003872
• 负责人: Arieh Warshel
• 金额: $ 33万
• 依托单位: University of Southern California
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0003872&HistoricalAwards=false
• 关键词: Structure; Function; Correlation; Proteins

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.

WARSHELMCB-0003872GTP结合蛋白在几乎所有生物体中的细胞功能和控制信号转导过程中的细胞功能中起着重要作用。 获得这些蛋白质作用的详细分子图片是对现代分子生物学的重要挑战。 G蛋白的研究经历了一项“结构革命”，其中G蛋白的新结构及其与相应的辅助蛋白和/或过渡状态类似物的复合物迅速出现。 这为阐明这些蛋白质作用的详细分子机制提供了独特的机会。在过去的几年中，开发了将酶结构与其功能相关联的计算机模拟方法。 发现这些方法在RAS催化的反应的研究中非常有用。 特别是，模拟表明GTP而不是GLN61是RAS酶机制的实际基础。 现在，这被广泛接受为G蛋白和相关系统作用的可能机制。 先前的研究还导致发现GTPase反应的速率常数和G磷酸的PKA之间发现了线性自由能关系（LFER）。 这项工作已作为基本建议提供了实验支持。 先前的研究表明，差距通过结合两种效应来运行。 首先，它通过主要使用Arg789的直接静电稳定来起作用。 其次，它通过“将” RAS“推到催化构型”而间接起作用。 这些仿真研究导致当前的项目利用了G蛋白结构研究的巨大进步。 （i）磷酸盐水解的潜在表面将得到验证和完善。 将比较计算出的和观察到的同位素效应和激活熵。 还将检查水辅助过渡状态的稳定性，包括相应的熵贡献。 这项工作将有助于解决机械问题和精炼EVB表面以进行G蛋白研究。（ii）将研究GAP的RAS激活，重点是GLN61和GLY12等突变的效果。 这些研究将检查这些残基是直接起作用还是通过稳定Ras的催化构型来起作用。这些蛋白质中GTPase反应的相对速率将被复制。这些研究将确定它们是否使用共同的机制。（iv）尽管G蛋白和TS类似物的复合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尚不清楚。 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提供的信息。