Quaternary Structure and Dynamics of Polydisperse Molecular Chaperone Complexes

合成后，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，这些蛋白质也起着至关重要的作用，通过保存和随后修复受损的蛋白质。这些蛋白质执行其“管家”职责的机制尚未完全了解，但该网络的故障导致各种疾病状态甚至过早死亡。在这个项目中，我们将集中在最不好理解的家庭的分子伴侣，小热休克蛋白（sHSPs）。除了它们的伴侣蛋白功能，这个蛋白质家族的成员已经被牵连在各种疾病状态，包括白内障，运动神经病，和阿尔茨海默氏病。具体来说，我们将研究如何sHSP行为和客户端蛋白在应激条件下相互作用，以及它们如何融入整个伴侣蛋白网络在细胞中。我们之前已经证明了sHSP是非常动态的，因为它们可以在快速的时间尺度上彼此交换亚基。它们倾向于同时填充一系列状态，并与其他sHSP共同组装，这阻碍了对其研究的常规方法。我们已经集中了我们以前的研究开发和采用质谱（MS）的策略，这些蛋白质的研究，并已开发出一个工具包，装备精良，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与解折叠靶蛋白之间形成的复合物，这些蛋白是sHSP伴侣途径中的关键分子，但人们对它们的了解仍然很少。我们的目标是研究它们的组织，动态波动，以及它们的形成和分解。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：气相中络合物的解离;实时反应监测;为了进一步探索sHSP和靶蛋白之间的这些复合物的分子细节，我们将进行分子生物学实验，其中我们特异性地突变sHSP和靶蛋白中的某些氨基酸。sHSP以模拟体内发现的修饰。因此，我们可以评估这些改变对它们与底物形成的复合物的结构和动力学的影响，使我们能够详细地探测sHSP活性的分子机制。因此，通过提供这样的洞察sHSP与解折叠蛋白的相互作用方式，我们将在某种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，并研究在模拟细胞应激条件下观察到的差异，我们希望获得对其功能机制的新见解。

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.