Solution NMR studies of the Hsp104/ClpB Chaperone

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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.