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.

几种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。其中包括阿尔茨海默氏症和帕金森氏病，II型糖尿病以及海绵状脑病，例如克鲁特兹菲尔德·贾科布病。为了制定有效的治疗策略以防止，减速或扭转这些疾病的进展，必须了解对触发和/或反向细胞内部蛋白质聚集的分子机制的理解。反过来，这是基于涉及聚集反转过程的分子玩家的结构和运动特性的详细定量表征。这样的玩家是HSP100/CLPB伴侣。这些可以被认为是由运动部件组成的微型分子机。了解这些机器的功能如何需要详细研究其三维结构以及这些结构如何随着时间的流逝而变化，因为机器在细胞中执行任务。 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对底物结合以及对不同核苷酸（例如ATP和ADP和（iv）的存在）的反应，以表征CLPB与参与分类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型分子机器能够分解蛋白质的机制，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NMR方法，这些方法将对一系列大分子应用产生广泛的影响，包括涉及通常较弱的分子相互作用并因此通过其他生物物理学方法重新固定的分子相互作用的应用。