Navigating the downhill protein folding regime via structural homologues.

Proteins that fold over free-energy barriers <or= 3RT are classified as downhill folders. This regime is characterized by equilibrium unfolding that is proportionally broader and more complex the lower the folding barrier. Downhill proteins are also expected to fold up in a few microseconds. However, the relationship between rate and equilibrium signatures is affected by other factors such as protein size and folding topology. Here we perform a direct comparison of the kinetics and equilibrium unfolding of two structural homologues: BBL and PDD. BBL folds-unfolds in just approximately 1 micros at 335 K and displays the equilibrium signatures expected for a protein at the bottom of the downhill folding regime. PDD, which has the same 3D structure and size, folds-unfolds approximately 8 times more slowly and, concomitantly, exhibits all the downhill equilibrium signatures to a lesser degree. Our results demonstrate that the equilibrium signatures of downhill folding are proportional to the changes in folding rate once structural and size-scaling effects are factored out. This conclusion has two important implications: (1) it confirms that the quantitative analysis of equilibrium experiments in ultrafast folding proteins does provide direct information about free-energy barriers, a result that is incompatible with the conventional view of protein folding as a highly activated process, and (2) it advocates for equilibrium-kinetic studies of homologous proteins as a powerful tool to navigate the downhill folding regime via comparative analysis. The latter should prove extremely useful for the investigation of sequence, functional, and evolutionary determinants of protein folding barriers.