The primary objective of this research proposal is to deconvolute the individual mechanistic steps that describe the interaction of the enzyme ClpA with its protein substrates. Green fluorescent protein targeted to ClpA by the 11-amino acid recognition tag, ssrA (GFP-ssrA) will be used as a model substrate.
The first aim of the proposed work is to determine the ratio of the work performed by the enzyme and the amount of energy released by ATP hydrolysis in the catalyzed reaction. This value will be reported as the energetic efficiency.
The second aim of the proposed work is to determine the contribution of individual subunits to the efficiency of substrate unfolding and transport.
The third aim of the proposed work is to determine the effect of substrate stability on the efficiency of protein unfolding by ClpA. Pre-steady state, single turnover kinetics (using stopped-flow and rapid chemical quench methods, if required), differential scanning calorimetry, and isothermal calorimetry will be used as the primary methods to obtain detailed mechanistic data. Steady-state (Michaelis-Menten) kinetic experiments will also be carried out to provide a point of comparison with pre-steady state data and with the data provided in the literature.
