title: A kinetic approach to determining the conformational stability of a protein that dimerizes after folding.
abstract: A strongly stabilized form of the β1 domain of the streptococcal protein G, termed Gβ1-M2, was previously obtained by an in vitro selection method for stabilized protein variants. It contains four substitutions, but how they contribute to the Gibbs free energy of denaturation (ΔG(D)) could not be determined, because, unlike the wild-type protein, Gβ1-M2 dimerizes in a spectroscopically silent reaction. Here we determined the ΔG(D) of the folded Gβ1-M2 monomer by using a kinetic approach that uncouples the folding of the monomer from dimerization. The conformational equilibration of the monomer is faster than dimer formation, and therefore, its stability constant could be determined from the ratio of the rate constants for monomer unfolding and refolding. In this approach, double-mixing experiments were essential for uncovering the unfolding kinetics of the transient Gβ1-M2 monomer and the association of the monomers after their folding. The analysis revealed that the selected substitutions stabilize the Gβ1-M2 monomer by 15 kJ mol(-1) in an additive fashion. The combination of single- and double-mixing kinetic experiments thus allowed us to determine the thermodynamic stability of a transient species that is inaccessible in equilibrium experiments. It can be applied for proteins in which monomer folding and oligomerization are kinetically uncoupled.
authors.affiliation: Laboratorium für Biochemie und Bayreuther Zentrum für Molekulare Biowissenschaften, Universität Bayreuth, D-95440 Bayreuth, Germany.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Hoffmann-Thoms
authors.forename: Franz-Xaver
authors.initials: FX
authors.lastname: Schmid
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000187:drug effects; D019791:Guanidine / Q000494:pharmacology; D007700:Kinetics; D008958:Models, Molecular; D011487:Protein Conformation; D017510:Protein Folding; D055503:Protein Multimerization; D055550:Protein Stability
chemical_list: D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus; D019791:Guanidine