Abstract
Most proteins have DnaK binding sites. DnaK is an E. coli Hsp70 molecular chaperone which helps prevent protein aggregation by assisting co- and post-translational protein folding. How extensively and by what mechanism does DnaK interact with the proteins? We know very little about this important question. We used RNase HD and SH3 as model protein substrates to study how DnaK (and its co-chaperones DnaJ and GrpE) interacts with nonobligatory clients (i.e. proteins capable of folding even without the assistance of chaperones) and to provide insights into mechanism of action of DnaK. Stopped-flow circular dichroism, size-exclusion chromatography and enzyme activity assays provide evidence for kinetic retardation of folding due to DnaK-substrate complex formation. Furthermore, multidimensional NMR and photo-CIDNP (photo-chemically induced dynamic nuclear polarization) provide atomic level details regarding DnaK-substrate interactions. Overall, a combination of various experimental techniques provides insights into how the DnaK chaperone assists protein folding within the cellular environment.
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