Solution NMR studies of the Hsp104/ClpB Chaperone

一些人类疾病是由蛋白质的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氏症和帕金森氏症，II型糖尿病和海绵状脑病，如克雅氏病。为了开发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的分子机制。这反过来又是基于对参与聚集逆转过程的分子参与者的结构和运动属性的详细的、定量的描述。Hsp100/ClpB就是这样一组玩家。这些可以被认为是由运动部件组成的微型分子机器。要了解这些机器是如何工作的，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，这些结构如何随着时间的推移而变化。因此，本建议的目标是使用溶液核磁共振(核磁共振)谱(I)绘制出Hsp100/ClpB机器内的重要运动，(Ii)在原子水平上建立伴侣与目标底物之间的相互作用位置，(Iii)确定Hsp100/ClpB分子机器的不同运动部分是如何相互组织的，以及它们的相对位置如何随着底物结合和不同核苷酸(如ATP和ADP)的存在而变化，以及(Iv)表征ClpB与参与解聚过程的其他分子分子之间的相互作用。这项拟议的研究将提供对大分子机器能够分解蛋白质的机制的详细见解，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，这些方法将对一系列大分子应用产生广泛影响，包括那些涉及分子相互作用的方法，这些分子相互作用往往很弱，因此无法通过其他生物物理方法进行详细研究。

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.