Transient Nonlinear Infrared Spectroscopy of Ubiquitin Unfolding Dynamics

Abstract

We have structurally resolved the nanosecond to millisecond unfolding of ubiquitin with transient amide I two dimensional infrared (2D IR) and dispersed vibrational echo (DVE) spectroscopy following variable temperature jumps. 2D IR and DVE, a measurement related to the 2D IR spectrum projected onto the ω3 axis, are nonlinear techniques capable of measuring secondary structure content with picosecond time resolution. The equilibrium 2D IR spectrum reveals features resulting from delocalized β-sheet vibrations with dipoles oriented parallel (ν‖) and perpendicular (ν⊥) to the strands. Transient 2D IR spectra show a blue shift of the ν⊥ vibration and disappearance of a cross peak between ν⊥ and ν‖ over μs to ms time scales. Diagonal peak intensities and homogeneous linewidths also indicate the melting away of sheet structure and the concomitant increased mobility of β-strand amide groups. These changes reflect the sequential unfolding of the β-sheet beginning with the labile strands III-V and followed by strands I-II. This pathway is confirmed through transient DVE of ubiquitin mutants, in which local mutations affect the timescales assigned to specific structures. The free energy landscape is evaluated through comparison of experiment and 2D IR spectra calculated from molecular dynamics simulations of ubiquitin unfolding using a structure-based model. The separation of timescales, stretched exponential relaxation, and probe-dependent response are consistent with the observation of μs downhill unfolding of a sub-ensemble that is prepared at the transition state followed by ms activated unfolding kinetics. The downhill unfolding is characterized through temperature jumps initiated and ending at variable temperatures. The increased downhill unfolding amplitudes and slowing timescales that accompany increases in temperature indicate that multiple unfolding pathways become accessible at higher temperatures.