Thermally induced early events of ribonuclease A under reducing conditions: Evidenced by principal component analysis and two-dimensional correlation infrared spectroscopy

The thermally induced unfolding of ribonuclease A in the presence of 2-mercaptoethanol has been followed by fluorescence spectroscopy and Fourier transform infrared (FTIR) spectroscopy. Principal component analysis (PCA) in combination with two-dimensional (2D) infrared correlation spectroscopy has been employed as an attractive method for the investigation of subtle pretransitional conformational changes in the protein. Separate analyses of different stages indicated that, in stage I the change of β1(43–49) and α2(24–34) might be the main process; in stage II, β1 was further unfolded, changes of α1(3–13) and another species of β2(61–63) and β4(79–87) involved. Furthermore, quantitative analysis of the power spectra extracted from the synchronous 2D contour maps revealed that the intensity variations from pretransitional stages (below 47 °C) were 5% less than that for the main unfolding. Meanwhile, under reducing conditions the early subtle structural changes occurred in a non-cooperative manner, in contrast to what was found under non-reducing conditions.