Reply to Campos et al.: Direct observation versus ambiguous kinetics and thermodynamics

Most conventional experiments cannot distinguish a multimodal distribution of separate states in fast exchange from a unimodal distribution having the mean properties of the multimodal components (1). The “wealth of available thermo dynamic and kinetic data” posited to support unimodal folding of BBL (2) support equally well or better a mechanism of ultra-fast folding of the protein with separate native (N) and denatured (D) states (ref. 3 and references therein). But, our observation of bimodal distributions in single-molecule FRET experiments on BBL clearly demonstrates the presence of discrete populations separated by an energy barrier and falsifies unimodal folding (4).