Pressure versus temperature unfolding of ribonuclease A: An FTIR spectroscopic characterization of 10 variants at the carboxy-terminal site

FTIR spectroscopy was used to characterize and compare the temperature- and pressure-induced unfolding of ribonuclease A and a set of its variants eng ineered in a hydrophobic region of the C-terminal part of the molecule post ulated as a CFIS. The results show for all the ribonucleases investigated, a cooperative, two-state, reversible unfolding transition using both pressu re and temperature. The relative stabilities, among the different sites and different variants at the same site, monitored either through the changes in the position of the maximum of the amide I' band and the tyrosine band, or the maximum of the band assigned to the beta -sheet structure, corrobora te the results of a previous study using fourth-derivative UV absorbance sp ectroscopy. In addition, variants at position 108 are the most critical for ribonuclease structure and stability. The V108G variant seems to present a greater conformational flexibility than the other variants. The pressure- and temperature-denaturated states of all the ribonucleases characterized r etained some secondary structure. However, their spectral maxims were cente red at different wavenumbers, which suggests that pressure- and temperature -denaturated states do not have the same structural characteristics. Nevert heless, there was close correlation between the pressure and temperature mi dpoint transition values for the whole series of protein variants, which in dicated a common tendency of stability toward pressure and heat.