SIDE-CHAIN MOBILITY OF THE BETA-LACTAMASE-A STATE PROBED BY ELECTRON-SPIN-RESONANCE SPECTROSCOPY

Beta-lactamase from Bacillus licheniformis forms a stable compact inte rmediate state at low pH and moderate salt concentration (the A state) , with properties consistent with a molten globule. A single cysteine residue was introduced into this class A beta-lactamase by site-direct ed mutagenesis at position 166. A spin label was attached to the thiol of this cysteine residue via a disulfide bond as a probe of the side- chain mobility. The mutant protein and tbe spin-labeled derivative exh ibited similar conformational properties to the wild-type enzyme at ac idic pH. The A state induced by chloride or trichloroacetate (TCA) ani ons was characterized by circular dichroism and esr. Tbe A state at pH 0.5 (0.32 M HCl), or at pH 2 in the presence of 8 mM TCA or 0.4 M Cl- , had comparable amounts of secondary structure to the native state bu t lacked significant tertiary structure, as judged by the lack of near -UV circular dichroism. Analysis of the esr spectral line widths showe d that the mobility of the spin label in the A state was similar to th at in the native state and much less mobile than in the unfolded state , indicating significant constraints on the side-chain mobility in thi s region of the molecule in the A state. Tbe implications of this find ing to the structure of the A state are discussed.