title: Salt-bridges can stabilize but do not accelerate the folding of the homodimeric coiled-coil peptide GCN4-p1.
abstract: Double mutant cycle analysis was employed to ascertain the role of intra- and interchain salt-bridges in the folding and stability of the dimeric coiled-coil peptide, GCN4-p1, the 33-residue leucine zipper domain of the transcriptional activator GCN4. Equilibrium circular dichroism studies of the urea-induced unfolding reaction at neutral pH revealed that both types of ionic interactions, localized primarily in the N-terminal portion of the molecule, enhance the stability of the native coiled-coil. By contrast, comparable stopped-flow circular dichroism studies indicate that the salt-bridge interactions, with one possible exception, are not well formed in the transition state for folding. Although the E22Q/R25A double mutant failed to fold, fragmentation studies suggest that the E22/R25 intramolecular salt-bridge may play a critical role in stabilizing C-terminal nascent helices that drive the association reaction. The remaining salt-bridges appear to stabilize the parallel-stranded coiled-coil architecture of GCN4-p1 only after the peptide traverses the rate-limiting, dimeric transition state.
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA.
authors.forename: Beatriz
authors.initials: B
authors.lastname: Ibarra-Molero
authors.forename: Jill A
authors.initials: JA
authors.lastname: Zitzewitz
authors.forename: C Robert
authors.initials: CR
authors.lastname: Matthews
mesh_terms: D019943:Amino Acid Substitution; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D019281:Dimerization; D007700:Kinetics; D011489:Protein Denaturation; D017510:Protein Folding; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics; D055672:Static Electricity; D040681:Structural Homology, Protein; D013816:Thermodynamics
chemical_list: D004268:DNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; D011494:Protein Kinases