Structure is lost incrementally during the unfolding of barstar

Coincidental equilibrium unfolding transitions observed by multiple structu ral probes are taken to justify the modeling of protein unfolding as a two- state, N reversible arrow U, cooperative process. However, for many of the large number of proteins that undergo apparently two-state equilibrium unfo lding reactions, folding intermediates are detected in kinetic experiments. The small protein barstar is one such protein. Here the two-state model fo r equilibrium unfolding has been critically evaluated in barstar by estimat ing the intramolecular distance distribution by time-resolved fluorescence resonance energy transfer (TR-FRET) methods, in which fluorescence decay ki netics are analyzed by the maximum entropy method (MEM). Using a mutant for m of barstar containing only Trp 53 as the fluorescence donor and a thionit robenzoic acid moiety attached to Cys 82 as the fluorescence acceptor, the distance between the donor and acceptor has been shown to increase incremen tally with increasing denaturant concentration. Although other probes, such as circular dichroism and fluorescence intensity, suggest that the labeled protein undergoes two-state equilibrium unfolding, the TR-FRET probe clear ly indicates multistate equilibrium unfolding. Native protein expands progr essively through a continuum of nativelike forms that achieve the dimension s of a molten globule whose heterogeneity creases with increasing denaturan t concentration and which appears to be separated from the unfolded ensembl e by a free energy barrier.