KINETIC CHARACTERIZATION OF THE CHEMOTACTIC PROTEIN FROM ESCHERICHIA-COLI, CHEY - KINETIC-ANALYSIS OF THE INVERSE HYDROPHOBIC EFFECT

CheY, the 129 amino acid chemotactic protein from Escherichia coil, is a good model for studying the folding process of the parallel alpha/b eta family of proteins. A study of the folding kinetics of CheY using fluorescence and far-UV circular dichroism (CD) stopped-flow measureme nts is reported. CheY has three prolines, two of them in the trans con formation and one, Pro110, with a cis Lys-Pro peptide bond. This prote in presents a unimolecular, but complex, kinetic mechanism that is dom inated by a slow phase compatible with a trans-cis isomerization. Muta tion of Pro110 to Gly results in the disappearance of this slow phase, indicating that this cis prolyl bond is responsible for it. The slow phase is catalyzed in a very inefficient way by prolyl isomerase, indi cating that the cis bond is poorly accessible to the enzyme during ref olding. In agreement with this is the fact that the isomerization of t he Lys109-Pro110 bond occurs in an intermediate which contains 96% of the native far-UV CD signal and 80% of the native fluorescence signal. Analysis of the unfolded protein with all its prolines in the native conformation shows the existence of a very stable intermediate in the folding reaction. Mutation of a hyperexposed hydrophobic residue, Phe1 4, to Asn results in an increase in the free energy of unfolding of th e protein of similar to 3 kcal mol(-1). Kinetic analysis of the unfold ing and refolding reactions of this mutant indicates that the major st abilization effect comes from the relative destabilization of the unfo lded state and the kinetic intermediate with respect to the transition state, providing kinetic evidence for the inverse hydrophobic effect. This could also indicate the existence of nonnative interactions in f olding intermediates.