Structure of functionally important dynamic states of the proteasome

描述(申请人提供)：蛋白酶体是以受调节的方式降解细胞内蛋白质的巨大分子复合体。它们由两个亚组分组成--一个19S调节颗粒(RP)和一个蛋白水解性20S催化颗粒(CP)。这样做的目的是 应用是了解与功能相关的蛋白酶体结构动力学。几个实验室的最新发现提供了蛋白酶体的亚纳米分辨率结构。然而，这些美丽的模型并不完整。首先，底物缺乏。在第一个具体目标中， 将决定蛋白质底物如何与蛋白酶体结合。这一目标将研究底物与三个关键的蛋白酶体作用部位之间的相互作用模式：底物结合、经历活性转位的延伸多肽以及折叠结构域解开时的收缩。其次，目前的模型是静态的。在第二个特定目标中，各种生化方法将被用来捕捉底物与蛋白酶体在一组与不同功能状态相关的特定构象状态中的结合。一个 被广泛接受但未经检验的范式是，蛋白质的展开和易位伴随着蛋白酶体的大规模构象变化。这一观点将通过确定在特定状态下底物处理的蛋白酶体的结构来检验。第三个具体目标将调查最近的生化观察的结构基础，证明了蛋白酶体CP内的同质变构。在生化调节和确定细胞如何在各种形式的经典和混合蛋白酶体之间分配CP池时，CP内的通信可能是重要的。用两个PI构建这一应用的基本原理是，实现这个项目的科学目标需要他们的持续互动，以开发和进一步开发源自每个申请者实验室的方法：能够快速高分辨率结构确定的新型低温EM方法，以及能够形成用于结构研究的稳定和可调的蛋白酶体底物复合体的新型蛋白酶体底物。拟议的研究将以原子水平的分辨率可视化功能相关的蛋白酶体复合体。蛋白酶体由三磷酸腺苷结合和水解提供能量，并利用由此产生的能量来移动和展开它们的底物。然而，将蛋白酶体功能与结构联系起来的信息很少。 动力学。这些研究的长期目标是了解蛋白酶体作用的生物力学。这项拟议的研究意义重大，因为它是将我们对蛋白酶体的理解从静态对象转变为动态参与者的第一步。

DESCRIPTION (provided by applicant): Proteasomes are giant molecular complexes that degrade intracellular proteins in a regulated manner. They are composed of two subassemblies- a 19S regulatory particle (RP) and a proteolytic 20S catalytic particle (CP). The objective of this application is to understand functionally relevant proteasome structural dynamics. Recent findings from several labs provide subnanometer resolution structures of the proteasome. These beautiful models, however, are incomplete. First, substrate is absent. In the first Specific Aim it will be determined how protein substrates engage the proteasome. This Aim will investigate the mode of interaction between substrate and three key proteasome sites of action: substrate binding, extended polypeptide undergoing active translocation and the constriction against which a folded domain unravels. Second, the present models are static. In the second Specific Aim various biochemical approaches will be used to trap substrate engagement with proteasome in a set of specific conformational states associated with distinct functional states. A widely accepted yet untested paradigm is that protein unfolding and translocation is coupled with large-scale conformational changes of the proteasome. This view will be tested by determining structures of proteasomes with substrate processing in specific states. The third Specific Aim will investigate the structural basis of recent biochemical observations demonstrating homotropic allostery within the proteasome CP. Communication within the CP may be important in biochemical regulation and in determining how the cell allocates the CP pool among various forms of classic and hybrid proteasomes. The rationale for structuring this application with two PIs is that accomplishing the scientific goals of this project requires their continuing interaction to exploit and further develop methods that originated in the labs of each of the applicants: Novel cryoEM methods, which enable rapid high-resolution structure determination, and novel proteasome substrates, which enable formation of stable and tunable proteasome-substrate complexes for structural studies. The proposed studies will visualize functionally relevant proteasome complexes at atomic- level resolution. Proteasomes are fueled by ATP binding and hydrolysis and use the energy so derived to move and unfold their substrates. However, little information is available that relates proteasome function to structural dynamics. The long term goal of these investigations is to understand the biomechanics of proteasome action. The proposed research is significant because it is a first step in transforming our understanding of proteasomes from static objects to dynamic players.