Directed Evolution of the Dnak/ClpB chaperone system - towards substrate specific optimization and complementation with orthologous systems

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Molecular chaperones are highly conserved among all organisms and orthologous proteins may be found among a wide range of species. Accordingly they fulfill general housekeeping tasks but also allow cells to remain viable at stress conditions (mainly heat). Their principle tasks apply to a wide range of substrate proteins and as a consequence they are not highly specialized towards certain substrates. Since chaperones are so highly conserved, components that have to interact to perform certain tasks can originate from different species (orthologous systems) and yet be functional in vitro. We want to exploit cases where orthologous systems do not appear to be functional to learn about specific properties that may have been adapted for the relevant organism. Firstly, chaperones from the DnaK system of Thermus thermophilus should be evolved to confer heat resistance to Escherichia coli strains that lack the corresponding component. Secondly, these chaperones should be tailored for improved folding assistance towards selcted target substrates. The underlying functional and structural reasons for the desired changes will the be analysed in detail with spectroscopic, kinetic and structural methods.