Achieving Secondary Structural Resolution in Kinetic Measurements of Protein Folding: A Case Study of the Folding Mechanism of Trp-cage

Protein folding kinetics are often measured by monitoring the change of a single spectroscopic signal, such as the fluorescence of an intrinsic fluorophore or the absorbance at a single frequency within an electronic or vibrational band of the protein backbone. While such an experimental strategy is easy to implement, the use of a single spectroscopic signal can leave important folding events undetected and overlooked. Herein, we demonstrate, using the mini-protein Trp-cage as an example, that the structural resolution of protein folding kinetics can be significantly improved when a multi-probe and multi-frequency approach is used, thus allowing a more complete understanding of the folding mechanism.