Understanding the relationship between protein structure and dynamics remains a central topic in biophysics. Structural fluctuations, disorder, and conformational rearrangements are key elements of protein function. Measuring protein dynamics requires methods that combine structural sensitivity with fast time resolution, which remain particularly challenging.
In this seminar I will describe a recently-developed toolkit that combines ultrafast two-dimensional infrared (2D IR) spectroscopy with site-specific isotope labeling, spectral simulations based on spectroscopic maps, and Markov state models derived from extensive MD simulations. These techniques provide a detailed view of the equilibrium conformational heterogeneity and folding pathways of NTL9, a 39-residue a/ß miniprotein. Spectra of the folded ensemble suggest that certain regions exhibit significant disorder and solvent penetration into the backbone, while others remain in rigid hydrogen bonded configurations. Temperature-jump folding experiments provide evidence for a well-defined folding mechanism initiated by the formation of the rigid a-helix followed by the collapse of the ß-sheet.
2DIR spectroscopy measures snapshots of protein conformational ensembles with subpicosecond time resolution. Amide-I vibrations, consisting of backbone C=O stretching modes, contain a wealth of structural information, and 2D IR offers superior structural sensitivity by spreading the spectral information onto two frequency axes to measure couplings between different vibrational modes.