Nanosecond dynamics of the single tryptophan reveals multi-state equilibrium unfolding of protein GB1

Unfolding of the immunoglobulin binding domain B1 of streptococcal protein G (GB1) was induced by guanidine hydrochloride (GdnHCl) and studied by circ ular dichroism, steady-state, and time-resolved fluorescence spectroscopy. The fluorescence methods employed the single tryptophan residue of GB1 as a n intrinsic reporter. While the transitions monitored by circular dichroism and steady-state fluorescence coincided with each other, the transitions f ollowed by dynamic fluorescence were markedly different. Specifically, fluo rescence anisotropy data showed that a relaxation spectrum of tryptophan co ntained a slow motion with relaxation times of 9 ns in the native state and 4 ns in the unfolded state in 6 M GdnHCl. At intermediate GdnHCl concentra tions of 3.8-4.2 M, however, the slow relaxation time increased to 18 ns. T he fast nanosecond motion had an average time of 0.8 ns and showed no depen dence on the formation of native structure. Overall, dynamic fluorescence r evealed two preliminary stages in GB1 folding, which are equated with the f ormation of local structure in the beta(3)-strand hairpin and the initial c ollapse. Both stages exist without alpha-helix formation, i.e., before the appearance of any ordered secondary structure detectable by circular dichro ism. Another stage in GB1 folding might exist at very low (similar to 1 M) GdnHCl concentrations.