Folding Kinetics of Small Proteins Revealed by Tryptophan-Cysteine Contact Formation Experiments

Spectroscopic probes sensitive to the formation intra-chain contacts are increasingly used to study the structural and dynamical properties of polypeptides. Quenching of the triplet state of tryptophan by close contact with cysteine enables the measure of contact formation rates without the need of extrinsic probes, thus being suitable for the study of natural proteins and peptides. We illustrate the use of this method to investigate the conformational dynamics of small two-state proteins and of beta-hairpin peptides in conditions close to native. The coexistence of folded and unfolded states can be revealed from the non-exponential relaxation of the excited triplet, enabling the characterization of both unfolded chain dynamics and folding kinetics. The comparison with protein fragments and model disordered peptides allows estimating the contribution of different chain regions to the folding process. Using this approach, we outline the kinetic pathway leading to the formation of the beta-hairpin structure of the C-terminal fragment of protein GB1, revealing the presence of misfolded states, as proposed in recent computational studies.