Effects of serine-to-cysteine mutations on b-lactamase folding

JAVIER SANTOS; VALERIA A. RISSO; MAURICIO P. SICA; MARIO R. ERMÁCORA

BIOPHYSICAL JOURNAL

Año: 2007 p. 1707 - 1718

0006-3495

B. licheniformis exo-small b-lactamase (ESBL) has two non sequential domains and a complex architecture. We replaced ESBL serine residues 126 and 265 with cysteine to probe the conformation of buried regions in each domain. Spectroscopic, hydrodynamic and chemical methods revealed that the mutations do not alter the native fold but distinctly change stability (S126C > wild-type >S126/265C> S265C ESBL) and the features of partially folded states. The observed wild-type ESBL equilibrium intermediate has decreased fluorescence but full secondary structure. S126C ESBL intermediate has the fluorescence of the unfolded state, no thiol reactivity, and partial secondary structure. S265C and S126/265C ESBL populate intermediate states unfolded by fluorescence and thiol reactivity but with full secondary structure. Mass analysis of S126/265C ESBL in the partially folded state proved that both thiol groups become exposed simultaneously. None of the intermediates is compatible with sequential domain unfolding. Molecular-dynamics simulation suggests that the stabilizing effect of the S126C substitution is due to optimization of van der Waals interactions and packing. On the other hand, destabilization induced by the S265C mutation results from alteration of the hydrogen bond network. The results illustrate the large impact that seemingly conservative serine-to-cysteine changes can have on the energy landscape of proteins.exo-small b-lactamase (ESBL) has two non sequential domains and a complex architecture. We replaced ESBL serine residues 126 and 265 with cysteine to probe the conformation of buried regions in each domain. Spectroscopic, hydrodynamic and chemical methods revealed that the mutations do not alter the native fold but distinctly change stability (S126C > wild-type >S126/265C> S265C ESBL) and the features of partially folded states. The observed wild-type ESBL equilibrium intermediate has decreased fluorescence but full secondary structure. S126C ESBL intermediate has the fluorescence of the unfolded state, no thiol reactivity, and partial secondary structure. S265C and S126/265C ESBL populate intermediate states unfolded by fluorescence and thiol reactivity but with full secondary structure. Mass analysis of S126/265C ESBL in the partially folded state proved that both thiol groups become exposed simultaneously. None of the intermediates is compatible with sequential domain unfolding. Molecular-dynamics simulation suggests that the stabilizing effect of the S126C substitution is due to optimization of van der Waals interactions and packing. On the other hand, destabilization induced by the S265C mutation results from alteration of the hydrogen bond network. The results illustrate the large impact that seemingly conservative serine-to-cysteine changes can have on the energy landscape of proteins.