Probing the structure of partially folded states of B. licheniformis beta-actamase by cysteine mutagenesis

RISSO, VA; SANTOS, J; SICA, MP; ERMÁCORA, , MR

Río de Janeiro, Brasil

Workshop; Workshop on Biocalorimetry and Biological Thermodynamics; 2006

The 265 residues protein B. licheniformis exo-small beta-lactamase (ESBL) has two nonsequential domains and a complex architecture. To study the conformation of partially folded states of ESBL, variants were prepared replacing serine at position 126 and/or 265 with cysteine, and so introducing thiols group in buried regions of each ESBL domain. All the ESBL variants fold to the native state. Thermal and urea-induced equilibrium unfolding of ESBL variants was monitored using optical and chemical probes. Replacement of the g oxygen of serine 126 and/or 265 has striking effects on the average conformation under partially denaturing conditions. Wild-type ESBL populates an intermediate state with slightly decreased fluorescence and full secondary structure. S126C is thermodinamically more stable than ESBL and partially unfolds to an intermediate state with no tertiary structure by tryptophanyl fluorescence, shields the thiol group from reacting with 5,5´-dithiobis-(nitrobenzoic acid) (DTNB), and possess partial secondary structure. S265C and S126/265C ESBL are less stable than ESBL and populate intermediate states that are unfolded by fluorescence and thiol reactivity but conserve full secondary structure. Mass analysis of S126/265C after chemical modification in the partially folded state proved that both thiol groups become exposed simultaneously. None of the intermediates are compatible with sequential unfolding of the ESBL domains. The results illustrate the complexity of the folding pathway of ESBL and reveal unsuspected differential effects between cysteine and serine that must be taken into account when using cysteine mutagenesis to study folding and also for protein engineering in general.