Solution NMR studies of interactions of proteins with molecular chaperones

新形成的蛋白质分子的成功折叠对其功能至关重要，而这反过来又是细胞生存所必需的。蛋白质分子在折叠途径上面临障碍，因为细胞非常拥挤的事实导致聚集或错误折叠的可能性很高。因此，细胞使用一系列辅助蛋白，称为分子伴侣，最大限度地减少蛋白质的错误折叠。这项研究计划的目的是开发和使用溶液核磁共振（NMR）光谱来研究这些分子伴侣和它们帮助折叠的多肽链之间的相互作用。具体来说，我们将针对最重要的分子伴侣系统之一，GroEL/GroES系统。(1)我们将探测GroEL/GroES的几个基板的结构和动力学，当这些基板在腔内。(2)将制备这些底物的折叠突变体，并在腔内和不存在分子伴侣的情况下进行研究，以深入了解底物和GroEL/GroES之间相互作用的性质。(3)将开发新的NMR方法来探测标记的底物分子和未标记的GroEL/GroES之间的相互作用，反之亦然。所描述的工作将提供深入了解蛋白质折叠的机制，以及大分子伴侣机器能够影响这一过程。此外，它将开辟研究不稳定蛋白质的可能性，目前无法通过使用伴侣来“保护”它们免于聚集。这将有助于研究“错误折叠”途径，其最终产物可导致与许多神经退行性疾病有关的纤维的形成。新的核磁共振方法将被开发出来，这将对一系列大分子应用产生广泛的影响。

The successful folding of newly formed protein molecules is essential for their function, which in turn is necessary for the survival of the cell. Protein molecules face obstacles on the folding pathway since the fact that the cell is very crowded leads to a high probability of aggregation or misfolding. The cell, therefore, uses a series of accessory proteins, termed molecular chaperones, which minimize protein misfolding. The objective of this research proposal is to develop and use solution Nuclear Magnetic Resonance (NMR) spectroscopy to study the interactions between these chaperones and the polypeptide chains that they help fold. Specifically, we will target one of the most important molecular chaperone systems, the GroEL/GroES system. (1) We will probe the structure and dynamics of several substrates of GroEL/GroES when these substrates are inside the cavity. (2) Folding mutants of these substrates will be prepared and studied inside the cavity and in the absence of chaperone to provide insight into the nature of the interactions between substrate and GroEL/GroES. (3) Novel NMR methods will be developed to probe interactions between labeled substrate molecules and unlabeled GroEL/GroES or vice versa. The work described will provide insight into the mechanism of protein folding and how large molecular chaperone machines are able to affect this process. Moreover, it will open up the possibility of studying unstable proteins that cannot be investigated currently by using the chaperone to 'protect' them from aggregation. This will facilitate the study of 'misfolding' pathways whose end-products can lead to the formation of fibers that have been implicated in a number of neurodegenerative diseases. New NMR methods will be developed that will have a broad impact on a range of macromolecular applications.