Title
β-sheet to α-helix conversion and thermal stability of β-Galactosidase encapsulated in a nanoporous silica gel
Author
M. Ines Burgos, Aylen Ochoa, María A. Perillo
Year
2019
Journal
Biochemical and Biophysical Research Communications
Abstract
The effect on protein conformation and thermal stability was studied for β-Galactosidase (β-Gal) encapsulated in the nanopores of a silicate matrix (Eβ-Gal). Circular dichroism spectra showed that, compared with the enzyme in buffer (Sβ-Gal), Eβ-Gal exhibited a higher content of α-helix structure. Heating Eβ-Gal up to 75 °C caused a decrease in the content of β-sheet structure and additional augments on Eβ-Gal components attributed to helical content, instead of the generalized loss of the ellipticity signal observed with Sβ-Gal. Steady state fluorescence spectroscopy analysis evidenced an Eβ-Gal structure less compact and more accessible to solvent and also less stable against temperature increase. While for Sβ-Gal the denaturation midpoint (Tm) was 59 °C, for Eβ-Galit was 48 °C. The enzymatic activity assays at increasing temperatures showed that in both conditions, the enzyme lost most of its hydrolytic activity against ONPG at temperatures above 65 °C and Eβ-Gal did it even at lower T values. Concluding, confinement in silica nanopores induced conformational changeson the tertiary/cuaternary structure of Eβ-Gal leading to the loss of thermal stability and enzymatic activity.
Instrument
J-810
Keywords
Circular dichroism, Secondary structure, Thermal stability, Protein folding, Biochemistry