A simple thermodynamic test to discriminate between two-state and downhill folding

The recent discovery of one-state folding, in which proteins unfold by progressive structural disorganizations (i.e., downhill folding), has emphasized the need for simple thermodynamic tests to discriminate between this behavior and classical two-state folding. On the basis of theoretical results from elementary statistical mechanical models, we propose such a test. The test involves monitoring the equilibrium unfolding transition induced by a combination of temperature and chemical denaturants with a probe that is sensitive to the average protein backbone conformation. The rationale is that the coupling between two different denaturation procedures can reveal subtle changes in protein conformational ensembles even when using bulk measurements. We demonstrate the applicability of the test by studying the unfolding process of the protein BBL, which has been previously characterized as a downhill folding protein. This test should be very useful for high-throughput design strategies and for the analysis of mutational effects in small proteins.