Two-dimensional infrared spectroscopy of the thermal unfolding of proteins

Steady-state and transient conformational changes upon the thermal unfolding of ubiquitin were investigated with femtosecond infrared spectroscopy of the amide I vibrations. Equilibrium temperature-dependent 2D IR spectroscopy reveals the unfolding of the $\beta $-sheet of ubiquitin through the loss of cross peaks formed between transitions arising from vibrations of the $\beta $-sheet. Transient unfolding following a nanosecond temperature jump is monitored with vibrational echo spectroscopy, a projection of the 2D IR spectrum. While the equilibrium study follows a simple two-state unfolding, the transient experiments observe complex relaxation behavior that differs for various spectral components and spans time scales from nanoseconds to milliseconds. By modeling the amide I vibrations of ubiquitin, this observation is explained as unfolding of the less stable strands \textit{III-V} of the $\beta $-sheet prior to unfolding of the hairpin that forms part of the hydrophobic core.