Daria Martin, Schering Stiftung Projektraum

 

Habilitation Prize 2002

Dr. Matthias Mayer

Center for Molecular Biology, University of Heidelberg

Mechanisms of Hsp70 mediated protein folding

Inside a living cell many polypeptides need the assistance of a set of specialized proteins, so called molecular chaperones, in order to attain and keep their active conformation. Central components of the cellular chaperone machinery are the 70 kDa heat shock proteins (Hsp70) that assist a large variety of folding processes including folding of newly synthesized and stress denatured polypeptides, translocation of proteins into organelles, assembly and disassembly of oligomeric complexes, and control of stability and activity of regulatory proteins.

The fundamental questions are how Hsp70 proteins in contrast to many other chaperones are able to refold misfolded proteins into the native state and what role ATP plays in this process.

To approach these questions we focused, firstly, on the ATPase cycle of Hsp70 chaperones and its regulation by co-chaperones and, secondly, on Hsp70-substrate interactions using the Escherichia coli Hsp70 homologue DnaK with its co-chaperones DnaJ and GrpE as major model system. We demonstrated that DnaJ together with a substrate protein synergistically stimulate the extremely low basal ATP hydrolysis rate of DnaK several thousand-fold. DnaJ, thereby, triggers the transition from the low affinity, ATP bound state of DnaK into the high affinity, ADP bound state leading to the trapping of the substrate. Since DnaJ can also bind substrate proteins, it acts as targeting factor.

The next question was how substrate release and thereby the half-live of the Hsp70-substrate complex is controlled. After substrate-trapping nucleotide exchange is limiting for the substrate release. We found that Hsp70 proteins differ in their nucleotide exchange rate by almost three orders of magnitude. Molecular modeling and mutational analysis lead to the identification of structural elements that are responsible for these differences. Since alteration of these structural elements in DnaK had profound effects on its chaperone activity, we proposed that variation of these elements was one of the mechanisms that allowed the evolutionary adaptation of the Hsp70 chaperones to the multitude of their functions.

A third question was how Hsp70 can bind many unfolded proteins promiscuously, but certain folded proteins with high specificity. The crystal structure of Hendrickson and coworkers and our biochemical data demonstrated that the affinity of DnaK for hydrophobic peptide stretches determines the efficiency with which DnaK refolds misfolded proteins. Such hydrophobic stretches are found usually in the interior of proteins and are only exposed upon unfolding explaining the promiscuous binding of misfolded proteins.

Furthermore, we identified two amino acid positions in the substrate binding domain of DnaK the alteration of which modulated the substrate specificity of DnaK. Interestingly, in the course of evolution these two sites are the only substrate contacting residues that were subjected to a considerable degree of variation suggesting adaptation to specific substrates and specific functions. We are currently further exploring the evolutionary plasticity of Hsp70 chaperones. We are also trying to elucidate how the substrate conformation is influenced by the binding of the chaperone.

2002_Mayer

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