Two-Dimensional Infrared Spectroscopy as a Probe of Protein Folding: Bridging the Gap between Experiment and Simulation

Two-dimensional infrared (2DIR) spectroscopy is a newly-developed experimental technique that measures protein structure and dynamics in solution with subpicosecond time resolution. Amide-I vibrations, consisting mainly of backbone C=O stretching modes, contain a wealth of structural information. Two-dimensional spectroscopy offers enhanced structural sensitivity by spreading the spectral information onto two frequency axes. Through a combination of temperature-jump 2DIR spectroscopy, isotope labeling, and Markov state models derived from molecular dynamics simulations, we develop a new method which can directly probe the structural rearrangements on timescales from nanoseconds to milliseconds. Markov state models provide an intuitive interpretation of the protein folding process while retaining much of the structural heterogeneity and diversity of folding pathways.The unfolding mechanism of a 39-residue α/β mini protein, NTL9, a two-state folder, is studied on timescales from 100 ns to 50 milliseconds. Transient 2DIR reveal a rapid sub-100 ns response that is attributed to weakening of the hydrogen-bonds, followed by unraveling of the beta sheet. The more stable helix is seen to denature on the 150 microsecond timescale. Experimental data is interpreted in the context of the recently-available Markov state model of NTL9. Simulated 2DIR spectra are generated for the structural ensemble, and are observed to be in great agreement with the temperature-jump 2DIR experiments. The results provide an elegant illustration of how a combination of cutting-edge experiments and state-of-the-art simulations gives new insights into the complex mechanism of protein folding.