Formation of a compact structured ensemble without fluorescence signature early during ubiquitin folding

The fluorescent Ub* mutant of the small globular protein ubiquitin is studied by multiple spectroscopic techniques under low temperature/high viscosity conditions to reveal unambiguously the formation of a compact structured intermediate preceding the barrier crossing to the native state. When detected by fluorescence intensity alone, ubiquitin appears to fold via a quasi two-state mechanism. In contrast, circular dichroism reveals the early formation of a structured ensemble, whose signal between 210 and 230 nm exceeds that of the native state, and whose unfolding curve is cooperative. The ensemble radius of gyration determined by small-angle X-ray scattering is only 1.2 times that of the native state, close to the classical molten globule value. Experiments performed in water/ethylene glycol mixtures at temperatures as low as -20 °C allow us to set an upper limit of ≤8k_BT on the barrier height for formation of the intermediate ensemble. The very small fluorescence burst phase indicates that the tryptophan residue is not yet packed into a nativelike conformation in the intermediate ensemble and that kinetics monitored by fluorescence intensity alone are not always a reliable indicator of two-state folding.