Reactivation of thermally inactivated pre-beta-lactamase by DnaK, DnaJ, and GrpE.
To understand the role of the 23-amino acid signal sequence in the folding and stability of beta-lactamase, the precursor and a mutant beta-lactamase with a 19-amino acid signal sequence deletion were synthesized in vitro using an Escherichia coli cell-free coupled transcription/translation system. Approximately 30% of the newly synthesized full-length precursor and 60% of the deletion mutant polypeptides were terminated and released from the ribosomes as active enzyme. Activity of the pre-beta-lactamase, but not the mutant, was unstable at 37 degrees C, suggesting that the signal sequence causes the enzyme to unfold. This inactivation was independent of ATP. Pre-beta-lactamase activity was stabilized by lowering the temperature to 30 degrees C. Furthermore, addition of the molecular chaperones DnaK/J and GrpE, in the presence of ATP and Mg2+, restored the activity of the temperature-inactivated precursor. The precursor formed a stable complex with DnaK and GrpE. Both ATP and DnaJ were required for recovery of enzymatic activity, indicating that DnaJ may bind transiently to the complex. These results suggest that the signal sequence of the pre-beta-lactamase causes a temperature-dependent unfolding of the synthesized enzyme and that DnaK/J and GrpE interact with unfolded pre-beta-lactamase to promote refolding of the protein into its native, enzymatically active conformation.
McCarthy
D
D
Department of Chemistry and Biochemistry, University of Texas at Austin, 78712, USA.
Kramer
G
G
Hardesty
B
B
D000255:Adenosine Triphosphate / Q000494:pharmacology; D001426:Bacterial Proteins / Q000378:metabolism*; D004792:Enzyme Precursors / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism*; D029968:Escherichia coli Proteins; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006358:Hot Temperature; D008274:Magnesium / Q000494:pharmacology; D013379:Substrate Specificity; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000096:biosynthesis / Q000378:metabolism*
D001426:Bacterial Proteins; C494635:DnaJ protein, E coli; D004792:Enzyme Precursors; D029968:Escherichia coli Proteins; C068113:GrpE protein, Bacteria; C489400:GrpE protein, E coli; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D065093:beta-Lactamase Inhibitors; D000255:Adenosine Triphosphate; C037640:pre-beta-lactamase; D001618:beta-Lactamases; C063842:dnaK protein, E coli; D008274:Magnesium