Solution NMR studies of interactions of proteins with molecular chaperones

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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.