The colicin E1 insertion-competent state: detection of structural changes using fluorescence resonance energy transfer.
The single cysteine residue (Cys-505) located in the hydrophobic membrane anchor domain in the colicin E1 COOH-terminal channel peptide was labeled with the thiol-specific fluorescent reagent IAEDANS [5-[[[(iodoacetyl)amino]ethyl]amino]naphthalene-1-sulfonic acid]. The labeling stoichiometry was nearly 1:1 [AEDANS: peptide (mol:mol)]. Eleven single Trp mutants of the channel peptide were prepared, and the FRET efficiency for each Trp residue (donor) and the AEDANS chromophore (acceptor), covalently attached to Cys-505, was measured. The FRET efficiencies for the various donor-acceptor pairs ranged from 15% to approximately 100% for the native peptide in solution (pH6.0). The FRET efficiency for the W-507 channel peptide-AEDANS adduct approached 100% since this adduct showed no detectable Trp fluorescence. Activation of the channel peptide to the insertion-competent state upon addition of the nonionic detergent octyl beta-D-glucoside [10,000:1 detergent: peptide (mol:mol)] resulted in decreased FRET efficiencies. The detergent-activated colicin E1 channel peptide-AEDANS adducts possessed significant in vitro channel activity at pH 6.0. The relative changes in the FRET efficiencies upon peptide activation ranged from -1% (W-495 channel peptide-AEDANS adduct) to 48% (W-355 channel peptide-AEDANS adduct). A direct correlation existed between the relative change in FRET efficiency upon channel peptide activation and the position of the Trp (donor) residue within the channel peptide primary sequence (higher relative delta E the closer the Trp donor was to the NH2 terminus), except for the W-484 channel peptide-AEDANS adduct, which showed a higher relative delta E than either W-443 or W-460 channel peptide-AEDANS adducts.(ABSTRACT TRUNCATED AT 250 WORDS)
Steer
B A
BA
Guelph-Waterloo Centre for Graduate Work in Chemistry, Department of Chemistry & Biochemistry, University of Guelph, Ontario, Canada.
Merrill
A R
AR
D000595:Amino Acid Sequence; D003087:Colicins / Q000737:chemistry*; D004735:Energy Transfer; D004926:Escherichia coli; D005456:Fluorescent Dyes; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009282:Naphthalenesulfonates; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry
D003087:Colicins; D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; C006421:1,5-I-AEDANS; D014364:Tryptophan