title: Characterization of an unfolding intermediate and kinetic analysis of guanidine hydrochloride-induced denaturation of the colicin E1 channel peptide.
abstract: The equilibrium unfolding pathway of the colicin E1 channel peptide was shown in a previous study to involve an unfolding intermediate, stable in approximately 4 M guanidine hydrochloride, which comprised primarily the C-terminal hydrophobic alpha-helical hairpin segment of the peptide [Steer, B. A., & Merrill, A. R. (1995) Biochemistry 34, 7225-7233]. In this study, the structural nature of this unfolding intermediate was investigated further, and it was found that the intermediate primarily consists of a dimer species and is comprised of two partially denatured monomeric peptides, which appear to be associated by hydrophobic interactions. The dimerized structure was detected by size-exclusion high-performance liquid chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, chemical cross-linking, and intermolecular fluorescence energy transfer. Using stopped-flow fluorescence spectroscopy, the kinetics of the denaturation and dimerization of the colicin E1 channel peptide in 4 M guanidine hydrochloride were examined. Denaturation kinetics were also investigated by wild-type peptide Trp fluorescence and 1-anilinonaphthalene-8-sulfonic acid binding. The kinetics of dimer formation were examined by monitoring the time dependence of intermolecular Trp to 5-[[2-[(iodoacetyl)amino]ethyl]amino]naphthalene-1-sulfonic acid fluorescence resonance energy transfer upon denaturation in 4 M guanidine hydrochloride. In addition, single Trp mutant peptides were employed as site-specific fluorescent probes of unfolding kinetics and reported diverse and characteristic unfolding kinetics. However, it was shown that following a rapid and major unfolding transition the peptide's core residues cluster slowly, by hydrophobic association, forming an intermediate species which is a prerequisite to dimerization. These equilibrium and kinetic unfolding data describe a unique unfolding mechanism where the channel peptide forms a partially unfolded dimerized structure in 4 M guanidine hydrochloride.
authors.affiliation: Department of Chemistry and Biochemistry, University of Guelph, Ontario, Canada.
authors.forename: B A
authors.initials: BA
authors.lastname: Steer
authors.forename: A R
authors.initials: AR
authors.lastname: Merrill
mesh_terms: D000817:Anilino Naphthalenesulfonates; D002850:Chromatography, Gel; D003087:Colicins / Q000737:chemistry* / Q000378:metabolism; D003432:Cross-Linking Reagents; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry*; D005456:Fluorescent Dyes; D019791:Guanidine; D006146:Guanidines; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D014364:Tryptophan
chemical_list: D000817:Anilino Naphthalenesulfonates; D003087:Colicins; D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; D006146:Guanidines; D008565:Membrane Proteins; C027132:1-anilino-8-naphthalenesulfonate; D014364:Tryptophan; D019791:Guanidine