Sulfate-Induced Effects in the On-Pathway Intermediate of the Bacterial Immunity Protein Im7*

摘要

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 Na_2SO_4 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 Na_2SO_4 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 Na_2SO_4 on the unfolding and refolding kinetics of Im7*,combined with detailed analysis of the resulting chevron plots,we show that decreasing the concentration of Na_2SO_4 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_I approx=0.8,beta_(TS2)approx=0.9 in 0 to 0.4 M Na_2SO_4).Analysis of 10 variants of the protein in 0.2 M Na_2SO_4 using OMICRON-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.