Equilibrium and kinetic analyses of unfolding and refolding for the conserved proline mutants of tryptophan synthase alpha subunit

Abstract

To elucidate the role of conserved proline residues of the tryptophan synthase alpha subunit from Escherichia coli in stability and folding, equilibrium and kinetic studies of the unfolding-refolding induced by guanidine hydrochloride for six mutant alpha subunits (Pro-->Ala) were carried out by peptidyl circular dichroism and aromatic fluorescence measurements at pH 7 and 25 degrees C. These results were analyzed assuming the presence of one intermediate (I) state in the denaturation process. (I) For all mutant and wild-type proteins, the Gibbs energy change (delta Gni(H2O)) in water between the native (N) and I states coincided with the difference (delta G++u(H2O)-delta G++r(H2O)) between the activation Gibbs energy changes in water for the unfolding (delta G++u(H2O) and refolding (delta G++r(H2O) reactions. This means that the early folding intermediate of the alpha subunit corresponds to the equilibrium intermediate. Delta Gni(H2O) values of all mutant proteins decreased compared with that of the wild-type protein. Gibbs energy change (delta Gid(H2O) in water between I and the denatured (D) states was not substantially affected by the substitutions. Delta G++u(H2O) and delta G++r(H2O) decreased and increased, respectively, for all mutant proteins. (2) Six conserved prolines played roles in stability and folding of the alpha subunit in a different manner: prolines 28 and 96 by stabilizing the N state and prolines 28, 96, 132, and 207 by destabilizing the I state. The contributions of prolines 57 and 62 to the stability were marginal. (3) Cis proline 28 was not the origin of the slow phase in the refolding kinetics assumed to arise from the cis-trans isomerization reaction of proline.