Thermodynamic and Hydrodynamic Examination of ClpB Assembly

Abstract

E. coli ClpB is a heat shock protein that belongs to the AAA+ protein family. Studies have shown that ClpB and its eukaryotic homologue, Hsp104, can disaggregate denatured proteins by themselves or cooperate with the DnaK chaperone system in vivo. It is thought that ClpB requires binding of nucleoside triphosphate to assemble into hexameric rings with protein binding activity and ClpB majorly exist as hexamer in the presence of nucleoside triphosphate. In contrast to this conclusion, our sedimentation velocity data show that ClpB can form hexamer in the absence of nucleotide and ClpB resides in a monomer-dimer-tetramer-hexamer equilibrium in the presence of ATPgS (a slowly hydrolysable ATP analog). ClpB hexamers exhibit fast subunits exchange in the absence of nucleoside triphosphate, while the exchange rates decrease in the presence of a large excess of ATPgS. We anticipate our studies on ClpB assembly to be a starting point of for understanding how ClpB hexamers disaggregate protein aggregates. For example, knowing the population of ClpB hexamers in solution is essential for the interpretation of it is only possible to study the energetics and kinetics data for the of ClpB catalyzed disaggregation processreaction by knowing the population of ClpB hexamer in solution.