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

lical structure in peptides and proteins has been extensively studied with circular dichroism (CD) spectroscopy. However, to date, complementary in-formation from infrared (IR) spectroscopy has not been reported. Using both IR and CD spectroscopy, we demonstrate here that the TFE-induced nonna-tive a-helical structure in two b-sheet-predominant proteins, b-lactoglobulin and a-chymotrypsin, is un-stable in comparison with those found in the a-helix-predominant proteins myoglobin and cytochrome c under identical conditions. IR spectra showed that, immediately after dissolution of the b-sheet proteins in 50 % (v/v) TFE, a strong amide I band component appears at 1654 cm21 in H2O and at 1650 cm 21 in D2O, which is ascribed to a-helical structure. However, the intensities of the a-helical bands decrease as a function of time, concomitant with the appearance of two new band components near 1620 and 1695 cm21 in H2O and 1612 and 1684 cm 21 in D2O, a typical IR spectral pattern for an intermolecular b-sheet aggregate. Clear gels begin to develop within 30 min. No similar spectral changes were observed for the a-helical proteins. CD spectra suggested initially that the TFE-induced a-helix was retained in the gelled state. However, further analysis of the spec-tra, and Gaussian function modeling with basic spectra, indicated that the apparent a-helix signal was actually due to a combination of signals from intermolecular b-sheet and residual a-helix. These results indicate that the TFE-induced nonnative a-helix structure in predominantly b-sheet proteins is unstable and readily converts to an intermolecu-lar b-sheet aggregate. © 1998 Academic Pres