Sulfate-induced effects in the on-pathway intermediate of the bacterial immunity protein Im7

Intermediates have now been identified in the folding, of a number of small, single-domain proteins. Here we describe experiments to determine the effect of Na2SO4 on the properties of the on-pathway intermediate formed early during the folding of the four-helical protein, Im7*. This intermediate, studied previously in 0.4 M Na2SO4, contains three of the four native helices and is fascinating in that several residues in helices I, II, and IV make non-native interactions that stabilize this state. Whether these contacts form as a consequence of the presence of Na2SO4, however, remained unresolved. Using kinetic analysis of the effect of Na2SO4 on the unfolding and refolding kinetics of Im7*, combined with detailed analysis of the resulting chevron plots, we show that decreasing the concentration of Na2SO4 from 0.4 to 0 M destabilizes the intermediate and rate-limiting transition (TS2) states by 7 and 10 kJ mol(-1), respectively, and has little effect on the relative compactness of these states compared with that of the unfolded ensemble (beta(1) approximate to 0.8, beta(TS2) approximate to 0.9 in 0 to 0.4 M Na2SO4). Analysis of 10 variants of the protein in 0.2 M Na2SO4 using Phi-values showed that the structural properties of the intermediate and TS2 are not altered significantly by the concentration of the kosmotrope. The data demonstrate that the rapid formation of a compact intermediate stabilized by non-native interactions during Im7* folding is not induced by high concentrations of the stabilizing salt, but is a generic feature of the folding of this protein.