A quantitative connection of experimental and simulated folding landscapes by vibrational spectroscopy

For small molecule reaction kinetics, computed reaction coordinates often mimic experimentally measured observables quite accurately. Although nowadays simulated and measured biomolecule kinetics can be compared on the same time scale, a gap between computed and experimental observables remains. Here we directly compared temperature-jump experiments and molecular dynamics simulations of protein folding dynamics using the same observable: the time-dependent infrared spectrum. We first measured the stability and folding kinetics of the fastest-folding β-protein, the GTT35 WW domain, using its structurally specific infrared spectrum. The relaxation dynamics of the peptide backbone, β-sheets, turn, and random coil were measured independently by probing the amide I′ region at different frequencies. Next, the amide I′ spectra along folding/unfolding molecular dynamics trajectories were simulated by accurate mixed quantum/classical calculations. The simulated time dependence and spectral amplitudes at the exact experimental probe frequencies provided relaxation and folding rates in agreement with experimental observations. The calculations validated by experiment yield direct structural evidence for a rate-limiting reaction step where an intermediate state with either the first or second hairpin is formed. We show how folding switches from a more homogeneous (apparent two-state) process at high temperature to a more heterogeneous process at low temperature, where different parts of the WW domain fold at different rates.