Two-dimensional correlation infrared spectroscopic study on the conformational changes occurring in the thermally induced pretransition of ribonuclease A

Thermal unfolding of ribonuclease A (RNase A) in deuterated Tris buffer is studied by Fourier transform infrared (FT-IR) spectroscopy. Two kinds of two-dimensional (2D) correlation spectroscopy, variable–variable (VV) 2D and sample–sample (SS) 2D spectroscopy, have been employed to analyze the observed thermally induced spectral variations of RNase A. Using SS 2D spectroscopy one can observe a pretransition at 45 °C prior to the main transition at 66 °C. This pretransition cannot be detected by a single-frequency analysis or the SS 2D correlation analysis of the original infrared spectra of RNase A. The VV 2D correlation spectroscopy study provides information about the structural changes that occur during these transitions: in the pretransition, the observed structural variations are associated with local conformational changes of an α-helical segment and the modification of a certain amount of β-sheet structure; in the main unfolding, changes in irregular and α-helical structures are followed by those in the β-sheet structure, including the antiparallel β-sheet components, resulting in the loss of secondary structure. This work demonstrates that 2D correlation spectroscopy can be used as an alternative to principal component analysis.