GroE-mediated folding of bacterial luciferases in vivo

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Abstract
In this study we present evidence indicating that GroE chaperonins mediate de novo protein folding of heterodimeric and monomeric luciferases under heat shock or sub-heat shock conditions in vivo. The effects of additional groESL and groEL genes on the bioluminescence of Escherichia coli cells expressing different bacterial luciferase genes at various temperatures were directly studied in cells growing in liquid culture. Data indicate that at 42° C GroESL chaperonins are required for the folding of the β subunit polypeptide of the heterodimeric αβ luciferase from the mesophilic bacterium Vibrio harveyi MAV (B392). In contrast, the small number of amino acid substitutions present in the luciferase β subunit polypeptide from the thermotolerant V. harveyi CTP5 suppresses this requirement for GroE chaperonins, and greatly reduces interaction between the β subunit polypeptide and GroEL chaperonin. In addition, GroESL are required for the de novo folding at 37° C of a MAV αβ luciferase fusion polypeptide that is functional as a monomer. No such requirement for luciferase activity is observed at that temperature with a fusion of the CTP5 α and β subunit polypeptides, although GroE chaperonins can still mediate folding of the CTP5 fusion luciferase. Bacterial luciferases provide a unique system for direct observation of the effects of GroE chaperonins on protein folding and enzyme assembly in living cells. Furthermore, they offer a sensitive and simple assay system for the identification of polypeptide domains required for GroEL protein binding.