title: Refolding kinetics of cytochrome c(551) reveals a mechanistic difference between urea and guanidine.
abstract: The energetic parameters for the folding of small globular proteins can be very different if derived from guanidine hydrochloride (GdnHCl) or urea denaturation experiments. A study of the equilibrium and kinetics of the refolding of wild-type (wt) cytochrome c(551) (cyt c(551)) from Pseudomonas aeruginosa and of two site-directed mutants (E70Q and E70V) shows that the nonionic nature of urea reveals the role of a salt bridge between residues E70 and K10 on the transition state, which is otherwise completely masked in GdnHCl experiments. Mixed denaturant refolding experiments allow us to conclude that the masking effect of GdnHCl is complete at fairly low GdnHCl concentrations ( congruent with 0.1 M). The fact that potassium chloride is unable to reproduce this quenching effect, together with the results obtained on the mutants, suggests a specific binding of the Gdn(+) cation, which involves the E70-K10 ion pair in wt cyt c(551). We propose, therefore, a simple kinetic test to obtain a mechanistic interpretation of nonlinear dependences of DeltaG(w) on GdnHCl concentration on the basis of kinetic refolding experiments in the presence of both denaturants.
authors.affiliation: Istituto Pasteur-Fondazione Cenci Bolognetti e Centro di Biologia Molecolare del CNR, Dipartimento di Scienze Biochimiche "A. Rossi Fanelli", Università di Roma "La Sapienza", 00185 Rome, Italy.
authors.forename: S
authors.initials: S
authors.lastname: Gianni
authors.forename: M
authors.initials: M
authors.lastname: Brunori
authors.forename: C
authors.initials: C
authors.lastname: Travaglini-Allocatelli
mesh_terms: D001426:Bacterial Proteins; D003574:Cytochrome c Group / Q000737:chemistry*; D019791:Guanidine / Q000737:chemistry*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D017510:Protein Folding; D011550:Pseudomonas aeruginosa / Q000737:chemistry; D014508:Urea / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D003574:Cytochrome c Group; D014508:Urea; C017167:cytochrome C(551); D019791:Guanidine