Sedimentation Equilibrium Analysis of ClpB Self-Association in Diluted and Crowded Solutions.

ClpB belongs to the Hsp100 family of ring-forming heat-shock proteins involved in degradation of unfolded/misfolded proteins and in reactivation of protein aggregates. ClpB monomers reversibly associate to form the hexameric molecular chaperone that, together with the DnaK system, has the ability to disaggregate stress-denatured proteins. Here, we summarize the use of sedimentation equilibrium approaches, complemented with sedimentation velocity and composition-gradient static light scattering measurements, to study the self-association properties of ClpB in dilute and crowded solutions. As the functional unit of ClpB is the hexamer, we study the effect of environmental factors, i.e., ionic strength and natural ligands, in the association equilibrium of ClpB as well as the role of the flexible N-terminal and M domains of the protein in the self-association process. The application of the nonideal sedimentation equilibrium technique to measure the effects of volume exclusion, reproducing in part the natural crowded conditions inside a cell, on the self-association and on the stability of the oligomeric species of the disaggregase will be described. Finally, the biochemical and physiological implications of these studies and future experimental challenges to eventually reconstitute minimal disaggregating machineries will be discussed.