Intermolecular β-Sheet Results from Trifluoroethanol-Induced Nonnative α-Helical Structure in β-Sheet Predominant Proteins: Infrared and Circular Dichroism Spectroscopic Study

2,2,2-Trifluoroethanol (TFE)-induced nonnative α-helical structure in peptides and proteins has been extensively studied with circular dichroism (CD) spectroscopy. However, to date, complementary information from infrared (IR) spectroscopy has not been reported. Using both IR and CD spectroscopy, we demonstrate here that the TFE-induced nonnative α-helical structure in two β-sheet-predominant proteins, β-lactoglobulin and α-chymotrypsin, is unstable in comparison with those found in the α-helix-predominant proteins myoglobin and cytochromecunder identical conditions. IR spectra showed that, immediately after dissolution of the β-sheet proteins in 50% (v/v) TFE, a strong amide I band component appears at 1654 cm−1in H2O and at 1650 cm−1in D2O, which is ascribed to α-helical structure. However, the intensities of the α-helical bands decrease as a function of time, concomitant with the appearance of two new band components near 1620 and 1695 cm−1in H2O and 1612 and 1684 cm−1in D2O, a typical IR spectral pattern for an intermolecular β-sheet aggregate. Clear gels begin to develop within 30 min. No similar spectral changes were observed for the α-helical proteins. CD spectra suggested initially that the TFE-induced α-helix was retained in the gelled state. However, further analysis of the spectra, and Gaussian function modeling with basic spectra, indicated that the apparent α-helix signal was actually due to a combination of signals from intermolecular β-sheet and residual α-helix. These results indicate that the TFE-induced nonnative α-helix structure in predominantly β-sheet proteins is unstable and readily converts to an intermolecular β-sheet aggregate.