Substrate Capture in Hsp70 Catalyzed by the Hsp40 J-domain

Tulane University, New Orleans LA

Investigators

Abstract

This research will analyze the role of the J-domain in the delivery of substrates by Escherichia coli Hsp40/DnaJ to Hsp70/DnaK. The Hsp40 and Hsp70 molecular chaperones cooperate in numerous cellular processes that require the modulation of protein-protein interactions. The Hsp40-Hsp70 chaperone machine dissociates protein complexes such as clathrin cages and transcriptional activation complexes. Hsp70 uses an ATPase activity to regulate substrate binding and dissociation. Hsp40 delivers substrate proteins to Hsp70 and stimulates the DnaK ATPase. In this research, the DnaJ-to-DnaK delivery process will be dissected using novel recombinant proteins composed of the J-domain fused to well-studied fluorescent peptide substrates. J-domain function is evident in the fact that the affinity of the fusion protein for DnaK increases in the presence of ATP. The affinities of proteins and peptides normally decrease in the presence of ATP unless DnaJ is present. Specifically, this research will investigate: (1) the influence of the J-domain on DnaK-binding affinity and kinetics of peptides and fusion proteins, (2) stimulation by the J-domain of the DnaK conformational change and ATPase activity, and (3) interaction of the J-domain- and peptide-binding sites on DnaK by NMR signal perturbation. This effort will illuminate the mechanism of Hsp40-Hsp70 cooperation. Molecular chaperones are machines that use the chemical energy of ATP to assemble and disassemble cellular structures. Some molecular chaperones act like conveyors, others like levers, and others like self-assembling scaffolds. The Hsp40-Hsp70 class molecular chaperone machine acts like a lever, using the energy of ATP to force protein subunits apart from each other. Whereas the Hsp70 component makes the force and acts on the protein subunits, the role of the Hsp40 component remains obscure. This research project will employ biophysical methods to analyze protein-protein binding, protein conformational changes, and ATP consumption to test the hypothesis that the Hsp40 acts like a fulcrum to couple the energy of ATP to lever action by the Hsp70. This work will have broad impacts on the advancement of biophysics, molecular machines, and the training of the next generation of scientists. Knowledge from this work will lead to molecular machines that create new materials and process information on the smallest-possible scale. Over the five-year project period, two Ph.D. students and at least five undergraduate students will work directly the project, and approximately twenty additional biophysics students will be exposed to the work through scientific exchanges within the Department of Biochemistry and the New Orleans Protein Folding Intergroup. Due to Tulane's location and ties to the community, approximately 20% of the trainees will be African-American.