Background: Bacteriophage fd is activated for infection by partial unfolding and prolyl isomerization. Results: NMR spectroscopy localized Pro-213-coupled unfolding to regions of the interdomain hinge of the phage gene-3-protein.Conclusion: Pro-213 regulates phage infectivity by a specific long-range effect on the conformational stability of the gene-3-protein.Significance: A proline switch controls the biological function in a remote part of a protein.
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.