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 Na(2)SO(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(2)SO(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 Na(2)SO(4), however, remained unresolved. Using kinetic analysis of the effect of Na(2)SO(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(2)SO(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) approximately 0.8, beta(TS2) approximately 0.9 in 0 to 0.4 M Na(2)SO(4)). Analysis of 10 variants of the protein in 0.2 M Na(2)SO(4) 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.
Astbury Centre for Structural Molecular Biology, Garstang Building, University of Leeds, UK.
Eva S
ES
Cobos
Sheena E
SE
Radford
D001426:Bacterial Proteins / Q000737:chemistry*; D002352:Carrier Proteins / Q000737:chemistry*; D003087:Colicins / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D007700:Kinetics; D017510:Protein Folding; D017433:Protein Structure, Secondary / Q000187:drug effects; D013431:Sulfates / Q000494:pharmacology*
D001426:Bacterial Proteins; D002352:Carrier Proteins; D003087:Colicins; C509253:E colicin-binding immunity protein Im7, E coli; D029968:Escherichia coli Proteins; D013431:Sulfates; C007104:colicin immunity proteins; C012036:sodium sulfate