IIBYT   23944
INSTITUTO DE INVESTIGACIONES BIOLOGICAS Y TECNOLOGICAS
Unidad Ejecutora - UE
artículos
Título:
beta-Galactosidase at the membrane-water interface. A case of an active enzyme with non-native conformation. Enviado para publicar.
Autor/es:
J. M. SANCHEZ; NOLAN, MV; M.A. PERILLO
Revista:
COLLOIDS AND SURFACES B-BIOINTERFACES
Editorial:
ELSEVIER SCIENCE BV
Referencias:
Lugar: Amsterdam; Año: 2013 vol. 108 p. 1 - 7
ISSN:
0927-7765
Resumen:
Previously we demonstrated that E.coli beta-galactosidase (β-Gal) binds to zwitterionic lipidmembranes improving its catalytic activity. To understand the activation mechanism from the protein perspective, here the thermal dependence of the catalytic activity was evaluated in conjunction with parameters derived from spectroscopy and calorimetry, in the presence and absence of egg-yolk phosphatidylcholine vesicles.In solution, the native state of β-Gal exhibits a loose conformation according to the λmax offluorescence emission which is in the upper end of the emission range for most proteins. A non-two state thermal unfolding mechanism was derived from DSC experiments and supported by the sequential unfolding temperatures exhibited by fluorescence (55ºC) and CD (60ºC) spectroscopies.Quenching of β-Gal´s intrinsic fluorescence, provided evidence for a novel and even looser folding for the lipid-bound protein. However, DSC data showed that the thermal unfolding in the presence of lipids tended to be displaced to higher temperatures compared to what happened in solution, with a significant decrease in ΔH, suggesting that only the population of bound protein molecules were involved in this process.Concluding, upon binding to a lipid-water interface β-Gal becames trapped in a partiallyunfolded state, more active and stable than that of the native protein in solution.