Molecular Mechanism of Protein Targeting by the Signal Recognition Particle

描述(由申请人提供)：蛋白质的正确定位对所有细胞都至关重要。信号识别颗粒(SRP)及其受体(SR)构成了将新合成的蛋白质运送到真核细胞内质网膜或细菌质膜的主要细胞机械。这一过程由SRP和SR中的两个同源GTP酶调控，这两个酶直接相互作用，使其对于机械性研究特别令人兴奋。尽管过去的工作已经定义了靶向途径的组成部分，但这一过程的分子机制仍不清楚。我们的总体目标是在生化和生物物理水平上破译这台普遍保守的靶向机器复杂的内部工作原理。我们的具体目标是了解SRP和SR GTP酶利用它们的GTP结合和水解循环来提供蛋白质靶向反应的空间和时间协调的机制。 为此，我们设想了三个具体的目标：(1)我们将定义和表征SRP-SR复合体形成过程中的动力学和构象中间产物及其相互的GTP酶激活；(2)我们将定义蛋白质靶向途径的其他组成部分-核糖体、信号序列和膜转位通道-是否、何时以及如何调节SRP和SR在结合和激活周期中的构象变化；(3)我们将使用一系列突变的GTP酶和GTP类似物来干扰SRP和SR的GTP酶循环中的特定构象步骤，并测试这些扰动如何影响蛋白质靶向反应中货物蛋白的识别、运输和卸载。这些实验将使我们能够确定每个GTP结合和水解事件在提供推动力或提高蛋白质靶向反应的保真度方面的确切作用。 最终，这些研究不仅将促进我们对蛋白质在细胞内定位过程的理解，而且还将在非常基本的水平上为分子识别和调控的一般原理提供新的见解。拟议的研究是最基本的性质，将有助于我们在分子水平上全面了解所有活细胞的生理学和病理学。

DESCRIPTION (provided by applicant): Proper localization of proteins is crucial to all cells. The signal recognition particle (SRP) and its receptor (SR) constitute the major cellular machinery that delivers newly synthesized proteins to the eukaryotic endoplasmic reticulum membrane, or the bacterial plasma membrane. This process is regulated by two homologous GTPases in the SRP and SR that directly interact with one another, making it particularly exciting for mechanistic investigations. Though past work has defined the components of the targeting pathway, the molecular mechanism of this process remains unclear. Our general goal is to decipher, at a biochemical and biophysical level, the intricate inner workings of this universally conserved targeting machine. Our specific goal is to understand the mechanism by which the SRP and SR GTPases use their cycles of GTP binding and hydrolysis to provide spatial and temporal coordination of the protein targeting reaction. To this end, three specific aims are envisioned: (1) We will define and characterize the dynamics and conformational intermediates during SRP-SR complex formation and their reciprocal GTPase activation; (2) We will define whether, when and how other components of the protein targeting pathway - the ribosome, the signal sequence, and the membrane translocation channel - modulate the conformational changes of SRP and SR during their binding and activation cycle; (3) We will use the array of mutant GTPases and GTP analogues to perturb specific conformational steps in the GTPase cycle of SRP and SR, and test how these perturbations affect the recognition, delivery and unloading of cargo protein during the protein targeting reaction. These experiments will allow us to define the precise role of each GTP binding and hydrolysis event in providing the driving force or improving the fidelity of the protein targeting reaction. Ultimately, these studies will not only advance our understanding of the process of protein localization within the cell, but also provide new insights into the general principles of molecular recognition and regulation at a very fundamental level. The proposed research is of a most basic nature, and will contribute profoundly to our general understanding of physiology and pathology of all living cells at the molecular level.