Experimental Studies on Protein Folding in the Presence of the Hsp70 Chaperone System

Abstract

The Hsp70 chaperone system (including DnaK, DnaJ and GrpE in bacteria) plays a vital role in preventing aggregation and assisting protein folding. While a lot of the chaperone biochemistry has already been worked out, very little is know about the interaction of DnaK and DnaJ with substrates. So far, such interaction had only been probed with small peptide or protein substrates incapable of independent/efficient refolding. This paucity of information has precluded studies on the competition between inter-molecular binding and intra-molecular folding. Such competition is important for cell viability because the accessibility of DnaK to substrate binding is modulated by the intrinsic rates of substrate folding/unfolding and by the substrate's thermodynamic stability.We developed a computational kinetic model to predict the interaction of protein substrates exhibiting two-state folding behavior with DnaK, DnaJ and GrpE. We found that, under physiological conditions, only proteins that fold slowly and/or have moderate thermodynamic stability are predicted to bind chaperones during their folding cycle.Experimental protein folding kinetics followed by stopped-flow in the presence and absence of the Hsp70 chaperone system shows good agreement with the predictions by the computational model. Furthermore, gel filtration and reverse phase chromatography data further support the stopped-flow results by providing evidence for DnaK-DnaJ-substrate interactions at equilibrium. This result is in agreement with the predictions of the computational model. In summary, the combination of experiments and computational predictions developed in this work is a powerful tool to help unveiling the relations between protein folding and chaperone binding.