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Author Santos, Javier ♦ Risso, Valeria A. ♦ Sica, Mauricio P. ♦ Ermácora, Mario R.
Source CiteSeerX
Content type Text
File Format PDF
Subject Domain (in DDC) Computer science, information & general works ♦ Data processing & computer science
Subject Keyword Serine-to-cysteine Mutation ♦ B-lactamase Folding ♦ Full Secondary Structure ♦ Thiol Reactivity ♦ Partial Secondary Structure ♦ S-265c Esbl ♦ Nonsequential Domain ♦ Licheniformis Exo-small B-lactamase ♦ Folded State ♦ Hydrogen-bond Network ♦ Sequential Domain Unfolding ♦ Esbl Populate Intermediate State ♦ Large Impact ♦ Buried Region ♦ Complex Architecture ♦ Mass Analysis ♦ Van Der Waals Interaction ♦ Unfolded State ♦ Native Fold ♦ Conservative Serine-to-cysteine Change ♦ Observed Wild-type Esbl Equilibrium Intermediate ♦ Thiol Group ♦ S-265c Mutation Result ♦ Esbl Serine ♦ Molecular Dynamic Simulation ♦ S-126c Substitution ♦ S-126c Esbl Intermediate ♦ Energy Landscape ♦ Chemical Method
Abstract ABSTRACT B. licheniformis exo-small b-lactamase (ESBL) has two nonsequential 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. Spec-troscopic, hydrodynamic, and chemical methods revealed that the mutations do not alter the native fold but distinctly change stability (S-126C. wild-type. S-126/265C. S-265C ESBL) and the features of partially folded states. The observed wild-type ESBL equilibrium intermediate has decreased fluorescence but full secondary structure. S-126C ESBL intermediate has the fluorescence of the unfolded state, no thiol reactivity, and partial secondary structure. S-265C and S-126/265C ESBL populate intermediate states unfolded by fluorescence and thiol reactivity but with full secondary structure. Mass analysis of S-126/265C 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 S-126C substitution is due to optimization of van der Waals interactions and packing. On the other hand, destabilization induced by the S-265C 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.
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Education Level UG and PG ♦ Career/Technical Study