Two-dimensional mid-IR and near-IR correlation spectra of ribonuclease A: Using overtones and combination modes to monitor changes in secondary structure

Abstract

We introduce near-IR spectroscopy as an ancillary tool for monitoring structural changes of proteins in aqueous solution using ribonuclease A (RNase A) as a model protein. The thermal unfolding of RNase A results in clear spectral changes in the near-IR and the mid-IR regions. In the near-IR the most pronounced changes are observed in the spectral region between 4820 and 4940 cm⁻¹. The strong NH combination band found at 4867 cm⁻¹ in the spectrum of native RNase A shifts to 4878 cm⁻¹ upon thermal unfolding. Hydrogen–deuterium exchange experiments that validate the NH character of this mode can also be used to estimate the number of unexchanged amide protons after exposure to D₂O. The transition profiles and temperatures derived from the temperature dependence of the NH combination mode were found to be practically identical with those derived from the temperature dependence of the CO amide I band in the mid-IR region, demonstrating that the near-IR region can be used as a conformation-sensitive monitor for the thermally induced unfolding of proteins in H₂O solution. A 2-dimensional correlation analysis was applied to the mid-IR and near-IR spectra of RNase A to establish correlations between IR bands in both regions. The correlation analysis demonstrates that the thermal unfolding of RNase A is not a completely cooperative process; rather it begins with some changes in β-sheet structure, followed by the loss of α-helical structures, and then ending with the unfolding of the remaining β-sheets. © 1998 John Wiley & Sons, Inc. Biospectroscopy 4: S19–S29, 1998