PRESSURE-INDUCED SECONDARY STRUCTURE CHANGES OF RIBONUCLEASE-A AND RIBONUCLEASE-S STUDIED BY FTIR SPECTROSCOPY

Fourier transform infrared (FTIR) spectroscopy combined with a resolution-enhancement technique has been used to characterize pressure-induced changes in the secondary structure of ribonuclease A (RNase A) and ribonuclease S (RNase S) in D2O solution at 30 degrees C. According to the observed changes in the amide I' band, both RNase A and RNase S are reversibly unfolded by application of high pressure. The pressure-unfolded state of the two proteins does not have any secondary structure elements characteristic of their native state, such as ct-helix, p-sheet, and reverse turn. However, there is a sight difference in the structural features between pressure-unfolded RNase A and RNase S, which is distinguished by the formation of nonnative turns only for RNase S. The dramatic structural transition of RNase A occurs over the pressure range between 570 and 1030 MPa. The pressure stability of RNase S is decreased by about 300 MPa compared with RNase A, despite little difference in the two native secondary structural features identified by FTIR spectroscopy. The structural features upon the pressure-induced unfolding are additionally characterized by the interesting behavior of hydrogen-deuterium exchange at high pressure. Most of the backbone amide protons protected at atmospheric pressure, which are involved in the alpha-helices and beta-sheet of both RNase A and RNase S, are exchanged with solvent deuterons in the pressure range where the two secondary structure elements are virtually identified as intact. It is suggested that the solvent accessibility to the internal regions is strongly correlated to the pressure stability. (C) 1995 John Wiley & Sons, Inc.