Generalized 2D and time-resolved FTIR studies of protein unfolding events

We present a combined two-dimensional (2D) IR and time-resolved IR study of unfolding events of proteins. The proteins that we selected for these experiments, the wild-type λ-Cro repressor and a designed variant, designated as Cro K56-[DGEVK], are known to undergo thermal unfolding through intermediates. The overall sequential order of the unfolding process of the wild-type λ-Cro repressor over the temperature range 20–55 °C was found to be α-helical prior to β-sheet structure. In contrast to that, the secondary structures of Cro K56-[DGEVK] appeared to change almost simultaneously to form the intermediate state at 65 °C. Thermal unfolding initiated by a rapid temperature-jump and monitored by time-resolved IR spectroscopy permitted us to follow unfolding reactions on the time-scale of 10−1 to 103 s. The analysis of the kinetic data of the wild-type λ-Cro repressor revealed that unfolding of the α-helices temporally proceeds before temporal changes in regions of its native β-sheet structure. In contrast to that, loss of the native β-sheet structure of the designed variant and unfolding of its α-helices was found to occur in concert.