The slow step of folding of Staphylococcus aureus PC1 beta-lactamase involves the collapse of a surface loop rate limited by the trans to cis isomerization of a non-proline peptide bond

We wished to test the hypothesis that the non proline cis to trans isomeriz ation of the peptide bond at position 167 in the S. aureus beta-lactamase P C1 exerts a significant controlling effect on the folding pathway of this e nzyme. The previous data presented in support of this hypothesis could not rule out the effect of factors unrelated to non-proline cis/trans isomeriza tion. We have used the plasmid pET9d to direct soluble overproduction of th e S. aureus beta-lactamase PC1 and a site-directed mutant (Ile 167 to Pro) in Escherichia coli, Following purification the proteins mere subjected to a comparative analysis of the kinetics of unfolding and refolding using the techniques of near- and far-UV circular dichroism spectroscopy and fluores cence spectroscopy in conjunction with "double-jump" experiments. Results s how that the fully-unfolded I167P mutant enzyme retains 20% of molecules in a fast-refolding form and that slower-refolding molecules fold faster than the recombinant wild-type enzyme. The final stage of folding involves fold ing of the Omega-loop into a conformation essential for enzymatic activity. In support of the original hypothesis, the folding of this Omega-loop is r ate limited by the isomerization of the Glu 166-Ile 167 peptide bond. (C) 1 998 Wiley-liss, Inc.