Microsecond protein folding through a compact transition state

Dynamic NMR methods have been employed to measure the folding and unfolding rate constants of two extremely fast-folding proteins. lambda(6-85), a truncated, monomeric form of the N-terminal domain of lambda repressor, refolds with a lifetime of approximately 250 mu s. These methods have also been applied to a thermostable lambda(6-85) variant with alanine substituted for glycine residues 46 and 48 in the third helix (G46A/G48A). Both proteins exhibit linear ln (k(f,u)) versus [urea] plots, consistent with two-state folding for both proteins. When extrapolated to OM urea, the data indicate that G46A/G48A folds with a lifetime of less than 20 mu s. The slopes of the In (k(f,u)) ver sus [urea] curves (m(u) and m(f)) indicate that the modest Gly --> Ala double mutation dramatically changes the transition state solvent accessibility. The transition state for lambda(6-85) has a fractional accessibility (m(u)(m(u) - m(f))) of 0.61, whereas the transition state for G46A/G48A is much more native-like, with a fractional accessibility of 0.16. The extraordinary change in the folding pathway that these mutations induce suggests that the intrinsic stability of helix 3 is an important determinant of the folding mechanism. (C) 1996 Academic Press Limited