Single amino acid mutations block a late step in the folding of β-lactamase from Staphylococcus aureus

Two single amino acid mutant proteins of β-lactamase PC1 from Staphylococcus aureus, P2 Thr40 → Ile and P54 Asp146 → Asn, have been investigated using urea-gradient polyacrylamide gel electrophoresis, circular dichroism and sedimentation velocity. Investigation of the folded states of the mutants has shown that compared to wild-type PC1 they are slightly more expanded, and have reduced aromatic circular dichroism, but the same content of secondary structure as PC1. The mutants exhibit fast refolding kinetics to the folded state, in contrast to PC1, which refolds only slowly. We conclude from these results that the folded mutants are in a state close to but distinct from the native state of PC1 and have certain properties in common with the compact intermediate in the folding of β-lactamase. Therefore, these single amino acid substitutions result in a folding pathway blocked at a point located after collapse of the already folded structural units into a globular shape, and close to the final reshuffling step that leads to the native state of the wild-type enzyme.