Guanidine hydrochloride-induced denaturation of the colicin E1 channel peptide: unfolding of local segments using genetically substituted tryptophan residues.
The soluble colicin E1 channel peptide has a roughly spherical, highly alpha-helical, compact structure. The structural unfolding properties of the colicin E1 channel peptide were analyzed using fluorescence techniques. The guanidine hydrochloride-induced unfolding pattern of the wild-type channel peptide was examined by monitoring intrinsic tryptophan fluorescence. Additionally, peptide unfolding was examined with the fluorophore, 1-anilinonaphthalene-8-sulfonic acid. In order to probe the unfolding of local segments, single-tryptophan channel peptides were constructed by site-directed mutagenesis. Shifts in fluorescence emission maxima of the single tryptophan residues were used to monitor site-specific unfolding events, in the presence of guanidine hydrochloride. The unfolding patterns reported by tryptophans in different regions of the peptide were diverse. The concentration of guanidine hydrochloride at the unfolding transition midpoint for each mutant peptide and the free energy of unfolding were calculated in order to estimate local segment stabilities. Also, secondary structure unfolding was monitored using circular dichroism spectroscopy. The results of unfolding analysis showed that the channel peptide's unfolding mechanism involves an intermediate structure stabilized by the C-terminal hydrophobic core of the peptide. Knowledge of the unfolding pattern of the soluble channel peptide will aid in the understanding of the secondary and tertiary structural interactions within the channel peptide and the mechanism of colicin E1 activation.
Steer
B A
BA
Guelph-Waterloo Centre for Graduate Work in Chemistry, Department of Chemistry and Biochemistry, University of Guelph, Ontario, Canada.
Merrill
A R
AR
D002942:Circular Dichroism; D003087:Colicins / Q000737:chemistry*; D019791:Guanidine; D006146:Guanidines / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry* / Q000235:genetics; D011489:Protein Denaturation; D017510:Protein Folding; D013861:Thiocyanates; D014364:Tryptophan / Q000737:chemistry* / Q000235:genetics
D003087:Colicins; D006146:Guanidines; D010455:Peptides; D013861:Thiocyanates; C054436:guanidine thiocyanate; D014364:Tryptophan; D019791:Guanidine