New light on protein folding: Unraveling folding and unfolding mechanisms using time-resolved and two-dimensional vibrational spectroscopy

How a protein folds from its one-dimensional sequence of amino acids into its three-dimensional, functional structure on biologically relevant time scales (typically on the micro- to millisecond time scale) is one of the most challenging questions currently investigated in several scientific disciplines and known as the protein-folding problem. This thesis describes experimental investigations of protein folding mechanisms using time-resolved and two-dimensional vibrational spectroscopy. The dynamical changes in the conformation of different, relatively small proteins and peptides were studied after a nanosecond perturbation of the folding⇋unfolding equilibrium using a laser-induced temperature-jump. The subsequent re-equilibration was probed by observing the time-dependent vibrational response in the amide I region using time-resolved infrared spectroscopy.
In this thesis, we show that salt-bridge interactions influence not only the folding equilibrium, but also strongly influence the folding kinetics of different α-helical model systems. This provides a possible explanation for the presence of apparently non-functional (because thermodynamically destabilizing) salt bridges in many biologically active proteins. We also have investigated the guanidinium-induced denaturation of a series of α-helical peptides with specifically tailored salt bridges, and found that one of the mechanisms underlying the protein-denaturing activity of guanidinium involves its competitive binding to carboxylate side groups involved in salt bridges that stabilize the native conformation. In addition, we show how transient infrared dispersed pump-probe spectroscopy can be applied to observe time-dependent 2D-IR cross peaks. Finally, we present a detailed investigation of the folding and unfolding mechanism of both the Trp-cage miniprotein and the zinc-finger ββα-motif FSD-1.