FOLDING KINETICS OF CHE-Y MUTANTS WITH ENHANCED NATIVE ALPHA-HELIX PROPENSITIES

in this work we study the folding kinetics of Che Y mutants in which t he helical propensity of each of its five alpha-helices has been great ly enhanced by local interactions (between residues close in sequence) . This constitutes an experimental test on the role of local interacti ons in protein folding, as well as providing new information on the de tails of the folding pathway of the protein Che Y. With respect to the first issue, our results show that the enhancement of helical propens ities by native-like local interactions in Che Y has the following gen eral effects: (1) the energetics of the whole Che Y folding energy lan dscape (folded state, intermediate, denatured state and main transitio n state) are affected by the enhancement of helical propensities, thus , native-like local interactions appear to have a low specificity for the native conformation; (2) our results support the idea, proposed fr om thermodynamic analysis of the mutants, that the denatured state und er native conditions becomes more compact upon enhancement of helical propensities; (3) the rate of folding in aqueous solution decreases in all the mutants, suggesting that the optimization of the folding rate in this protein requires low secondary structure propensities. Regard ing the description of the folding pathway of Che Y, we find evidence that the folding transition state of Che Y is constituted by two sub-d omains with different degree of helical structure. The first includes helices 1 and 2 which are rather structured, while the second encompas ses the last three helices, which are very unstructured. On the other hand, the same analysis for the folding intermediate indicates that al l the five alpha-helices are, on average, rather structured. Thus, sug gesting that a large structural reorganization of the last three alpha -helices must take place before folding can be completed. This conclus ion indicates that the folding intermediate of Che Y is a misfolded sp ecies. (C) 1997 Academic Press Limited.