AN EARLY INTERMEDIATE IN THE FOLDING REACTION OF THE B1 DOMAIN OF PROTEIN-G CONTAINS A NATIVE-LIKE CORE

The folding kinetics of a 57-residue IgG binding domain of streptococc al protein G has been studied under varying solvent conditions, using stopped-flow fluorescence methods, Although GB1 has been cited as an e xample of a protein that obeys a two-state folding mechanism, the foll owing kinetic observations suggest the presence of an early folding in termediate. Under stabilizing conditions (low denaturant concentration s, especially in the presence of sodium sulfate), the kinetics of fold ing shows evidence of a major unresolved fluorescence change during th e 1.5 ms dead time of the stopped-flow experiment (burst phase). Toget her with some curvature in the rate profile for the single observable folding phase, this provides clear evidence of the rapid formation of compact states with native-like fluorescence for the single tryptophan at position 43. In refolding experiments at increasing denaturant con centrations, the amplitude of the sub-millisecond phase decreases shar ply and the corresponding slope (m value) is only about 30% lower than that of the equilibrium unfolding curve indicative of a preequilibriu m transition involving cooperative unfolding of an ensemble of compact intermediates. The dependence on guanidine hydrochloride concentratio n of both rates and amplitudes (including the equilibrium transition) is described quantitatively by a sequential three-state mechanism, U r eversible arrow I reversible arrow N, where an intermediate (I) in rap id equilibrium with the unfolded state (U) precedes the rate-limiting formation of the native state (N). A 66-residue fragment of GB1 with a n N-terminal extension containing five apolar side chains exhibits thr ee-state kinetic behavior virtually identical to that of the 57-residu e fragment, This is consistent with the presence of a well-shielded na tive-like core excluding the N-terminal tail in the early folding inte rmediate and argues against a mechanism involving random hydrophobic c ollapse, which would predict a correlation between overall hydrophobic ity and stability of compact states.