Entrapping intermediates of thermal aggregation in alpha-helical proteins with low concentration of guanidine hydrochloride

Aggregation of proteins is a problem with serious medical implications and economic importance. To develop strategies for preventing aggregation, the mechanism(s) and pathways by which proteins aggregate must be characterized . In this study, the thermally induced aggregation processes of three alpha -helix proteins (myoglobin, cytochrome c, and lysozyme) in the presence and absence of 1.0 M guanidine hydrochloride (GdnHCl) were investigated by mea ns of infrared spectroscopy. In the absence of GdnHCl, intensities of the a lpha-helix bands (similar to 1656 cm(-1)) decrease as a function of tempera ture at above 50 degrees C. With myoglobin and cytochrome c, the loss of he lix bands was accompanied by the appearance of two new bands at 1694 and 16 23 cm(-1), indicative of the formation of intermolecular beta-sheet aggrega tes. For lysozyme, bands indicative of intermolecular beta-sheet aggregates did not appear in any significant intensity. In the presence of 1.0 M GdnH Cl, two major intermediate states rich in 3(10)-helix (represented by the b and at 1663 cm(-1)) and beta-turn structure (represented by the band at cm 1667 cm(-1)), respectively, were observed. These findings demonstrated that IR spectroscopic studies of protein aggregation using a combination of the rmal and chemical denaturing factors could provide a means to populate and characterize aggregation intermediates.