Solution NMR studies of the Hsp104/ClpB Chaperone

Hsp104/ClpB 分子伴侣的溶液 NMR 研究

• 批准号: RGPIN-2015-04347
• 负责人: Kay, Lewis
• 金额: $ 7.79万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=708475
• 关键词: Solution; NMR; studies; Hsp104; ClpB

Several human diseases are caused by protein misfolding and the accumulation of toxic protein aggregates. These include Alzheimer's and Parkinson's diseases, type II diabetes and the spongiform encephalopathies such as Creutzfeldt-Jakob disease. In order to develop effective therapeutic strategies to prevent, slow-down or reverse the progression of these diseases, an understanding of the molecular mechanisms involved in triggering and/or reversing protein aggregation inside the cell must be achieved. This, in turn, is predicated on a detailed, quantitative characterization of the structural and motional properties of the molecular players that are involved in the aggregation reversal process. One such set of players is the Hsp100/ClpB chaperones. These can be thought of as miniature molecular machines that are composed of moving parts. Understanding how these machines function requires a detailed study of their three-dimensional structures and how these structures change over time, as the machines carry out their tasks in the cell. The goal of the present proposal is, therefore, to use solution Nuclear Magnetic Resonance (NMR) Spectroscopy (i) to map out the important motions within the Hsp100/ClpB machine, (ii) to establish at atomic level resolution the sites of interaction between chaperone and target substrates, (iii) to determine how the different moving parts of the Hsp100/ClpB molecular machine are organized relative to each other and how their relative positions change in response both to substrate binding and to the presence of different nucleotides such as ATP and ADP and (iv) to characterize interactions between ClpB and other molecular players involved in the disaggregation process. The proposed research will provide detailed insight into the mechanisms by which large molecular machines are able to disaggregate proteins, ultimately contributing to the prevention of neurodegenerative disease. During the course of the proposed study, new NMR methods will be developed that will have a broad impact on a range of macromolecular applications, including those that involve molecular interactions that are often weak and hence recalcitrant to detailed studies by other biophysical approaches.

一些人类疾病是由蛋白质错误折叠和有毒蛋白质聚集体的积累引起的。这些疾病包括阿尔茨海默病和帕金森病、II 型糖尿病和海绵状脑病，例如克雅氏病。为了制定有效的治疗策略来预防、减缓或逆转这些疾病的进展，必须了解触发和/或逆转细胞内蛋白质聚集所涉及的分子机制。反过来，这又基于对参与聚集逆转过程的分子参与者的结构和运动特性的详细、定量表征。 Hsp100/ClpB 伴侣就是这样一组参与者。它们可以被认为是由运动部件组成的微型分子机器。要了解这些机器的功能，需要详细研究它们的三维结构，以及当机器在细胞中执行任务时这些结构如何随时间变化。因此，本提案的目标是使用解决方案核磁共振 (NMR) 光谱 (i) 绘制 Hsp100/ClpB 机器内的重要运动，(ii) 在原子级分辨率下建立相互作用位点分子伴侣和目标底物，(iii) 确定 Hsp100/ClpB 分子机器的不同移动部分如何相对于彼此组织，以及它们的相对位置如何响应底物结合和存在而变化不同的核苷酸，如 ATP 和 ADP，以及 (iv) 表征 ClpB 和参与解聚过程的其他分子参与者之间的相互作用。拟议的研究将详细了解大分子机器分解蛋白质的机制，最终有助于预防神经退行性疾病。在拟议的研究过程中，将开发新的核磁共振方法，这将对一系列大分子应用产生广泛的影响，包括那些涉及分子相互作用的方法，这些相互作用通常很弱，因此难以通过其他生物物理方法进行详细研究。