In situ observation of pressure-induced increased thermostability of two β-galactosidases with FT-IR spectroscopy in the diamond anvil cell

Abstract

We have previously reported that high hydrostatic pressure protected two β-galactosidases against thermal inactivation. Here, the Fourier transform infrared spectroscopy (FT-IR) technique was used to examine the pressure- and temperature-induced changes occurring in the structure of these two proteins. A thermostated diamond anvil cell allowed to follow in situ conformational changes against the variation of both temperature and pressure. FT-IR measurements have been carried out on proteins in D 2O-based solutions. Using D 2O, the amide I′ band (N-deuterated) can be measured without solvent interference. Analysis of the reduction in amide II intensities, which is due to peptide H–D exchange upon heating of the Aspergillus oryzae β-galactosidases in D 2O, showed that the temperatures for which H–D exchange acceleration occurred were 55, 65 and 60 °C at atmospheric pressure (0.1 MPa), 285 and 430 MPa, respectively. With the Escherichia coli β-galactosidase, these temperatures were 49, 52 and 51 °C at 0.1, 230 and 410 MPa, respectively. Interestingly, for the two enzymes considered, these values correlate quite well with the temperatures for which an irreversible activity loss of 50% is observed following a 30 min incubation. The pressure- and temperature-induced spectral changes in the amide I/I′ vibrational band of these two proteins are also discussed.