Folding Dynamics of the Trp-Cage Miniprotein: Evidence for a Native-Like Intermediate from Combined Time-Resolved Vibrational Spectroscopy and Molecular Dynamics Simulations

Trp-cage is a synthetic 20-residue miniprotein which folds rapidly and spontaneously to a well-defined globular structure more typical of larger proteins. Due to its small size and fast folding, it is an ideal model system for experimental and theoretical investigations of protein folding mechanisms. However, Trp-cage’s exact folding mechanism is still a matter of debate. Here we investigate Trp-cage’s relaxation dynamics in the amide I′ spectral region (1530–1700 cm–1) using time-resolved infrared spectroscopy. Residue-specific information was obtained by incorporating an isotopic label (13C═18O) into the amide carbonyl group of residue Gly11, thereby spectrally isolating an individual 310-helical residue. The folding–unfolding equilibrium is perturbed using a nanosecond temperature-jump (T-jump), and the subsequent re-equilibration is probed by observing the time-dependent vibrational response in the amide I′ region. We observe bimodal relaxation kinetics with time constants of 100 ± 10 and 770 ± 40 ns ...