Quaternary Structure and Dynamics of Polydisperse Molecular Chaperone Complexes

蛋白质合成后，将蛋白质折叠成正确的功能排列通常需要“分子伴侣”的帮助。当细胞受到压力时，这些蛋白质还通过保存和随后修复受损的蛋白质发挥至关重要的作用。这些蛋白质执行“家务”职责的机制尚不完全清楚，但这一网络的故障会导致多种疾病状态，甚至过早死亡。在这个项目中，我们将重点研究分子伴侣中最不为人所知的家族--小分子热休克蛋白(SHSPs)。除了伴侣功能外，这个蛋白家族的成员还参与了各种疾病，包括白内障、运动神经病和阿尔茨海默病。具体地说，我们将研究sHSP在应激条件下如何表现并与客户蛋白相互作用，以及它们如何融入细胞中的整个伴侣网络。我们之前已经展示了sHSP是非常动态的，因为它们可以在快速的时间尺度上相互交换亚基。他们倾向于同时填充一系列州，并与其他sHSP共同组装，这阻碍了他们研究的传统方法。我们以前的大部分研究都集中在开发和使用质谱学(MS)策略来研究这些蛋白质，并开发了一个装备良好的工具包来探测它们的结构和动力学。该项目的主要目标是详细研究sHSP和展开的目标蛋白之间形成的复合体，这些蛋白是SHSP伴侣途径中的关键分子，但仍然知之甚少。我们的目标是描述它们的组织、动态波动以及它们的形成和拆解。为了实现这一雄心勃勃的目标，我们将采用并同时进一步开发各种基于MS的方法，包括：气相中复合体的解离；实时反应监测；以及直接测量其大小。为了进一步探索SHSP与目标蛋白质之间的这些复合体的分子细节，我们将进行分子生物学实验，我们特异性地突变sHSP中的某些氨基酸以模拟体内的修饰。因此，我们可以评估这些变化对它们与底物形成的复合体的结构和动力学的影响，从而使我们能够详细地探索SHSP活性的分子机制。因此，通过对sHSPs与未折叠蛋白质相互作用的深入了解，我们将在一定程度上理解其伴侣功能的途径。此外，通过阐明它们在整个分子伴侣网络中的作用，并检查在模拟细胞应激的条件下观察到的差异，我们希望对它们的功能机制有新的见解。

After their synthesis, the folding of proteins into their correct functional arrangements often requires assistance by 'molecular chaperones'. These proteins also play a vital role when the cell is stressed, by preserving and subsequently repairing damaged proteins. The mechanism by which these proteins carry out their 'housekeeping' duties is not fully understood, yet malfunctioning of this network results in a variety of disease states and even premature death. In this project we will focus on the least well understood family of the molecular chaperones, the Small Heat-Shock Proteins (sHSPs). In addition to their chaperone function, members of this protein family have been implicated in various disease states including cataract, motor neuropathy, and Alzheimer's disease.Specifically, we will examine how sHSPs behave and interact with client proteins under stress conditions, and how they fit into the overall chaperone network in the cell. We have previously shown the sHSPs to be very dynamic, in that they can exchange subunits between each other on a rapid timescale. Their tendency to populate a range of states simultaneously, and to co-assemble with other sHSPs, has hampered conventional approaches to their study. We have focussed much of our previous research on developing and employing mass spectrometry (MS) strategies for the study of these proteins, and have developed a tool-kit well equipped for probing both their structure and dynamics. The primary goal of this project is to examine in detail the complexes formed between the sHSPs and unfolding target proteins, which are the pivotal molecules in the sHSP chaperone pathway yet remain very poorly understood. We aim to characterise their organization, dynamic fluctuations, and their formation and disassembly. To achieve this ambitious target we will employ, and concomitantly develop further, a variety of MS-based approaches, including: the dissociation of the complexes in the gas phase; real-time reaction monitoring; and the direct measurement of their size.To probe further the molecular details of these complexes between sHSP and target proteins we will perform molecular biology experiments in which we specifically mutate certain amino acids in the sHSPs to mimic modifications found in vivo. Thereby we can assess the effect of these alterations on the structure and dynamics of the complexes they form with substrate, allowing us to probe in detail the molecular mechanism of sHSP activity.Therefore, through providing such insight into the way sHSPs interact with unfolding proteins we will go some way to understanding the pathway of their chaperone function. Furthermore, by clarifying their role in the overall molecular chaperone network, and examining the differences observed under conditions mimicking cellular stress, we hope to gain novel insight into the mechanism of their function.