Protein Science, Vol 7, Issue 5 1164-1171, Copyright © 1998 by Cold Spring Harbor Laboratory Press

ARTICLE

Reactivation of thermally inactivated pre-{beta}-lactamase by DnaK, DnaJ, and GrpE

D. MCCARTHY, G. KRAMER and B. HARDESTY
Department of Chemistry and Biochemistry, University of Texas at Austin, Austin, Texas 78712

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{deg}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{deg}C. Furthermore, addition of the molecular chaperones DnaK/J and GrpE, in the presence of ATP and Mg(2+), 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.
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