Evidence for the amphipathic nature and tilted topology of helices 4 and 5 in the closed state of the colicin E1 channel.
The membrane-bound closed channel structure of helices 4 and 5 (Lys-406-Asp-446) of the colicin E1 channel domain was investigated by using a site-directed fluorescence labeling technique. A bimane probe was covalently attached to a cysteine residue in a series of single-cysteine mutant proteins to scan each helix in a residue-by-residue fashion. A variety of fluorescence properties of the bimane fluorophore were measured for both the soluble and membrane-bound states of the channel peptide, including the fluorescence emission maximum, fluorescence anisotropy, and membrane bilayer penetration depth. The fluorescence properties were collated for construction of a membrane-bound topology model of helices 4 and 5 of the channel domain. Finally, the data reveal that both helices 4 and 5 are two separate amphipathic alpha-helices that are situated parallel to the membrane surface. Dual fluorescence quencher analysis shows that helix 4 adopts a tilted topology in which its C-terminus is more buried than its N-terminus within the bilayer. In contrast, helix 5 is relatively solvent-exposed and located parallel to the interfacial region of the membrane surface. However, the loop region of both helices 4 and 5 was shown to be relatively buried within the bilayer. In addition, a least-squares fit of the data to a harmonic wave function indicated that both periodicity and frequency are typical for an amphipathic alpha-helix (3.75 +/- 0.1 residues per turn for helix 4 and 3.70 +/- 0.1 residues per turn for helix 5). Squared residual plots of the harmonic wave function also reveal that the N-terminus of helix 4 is elongated by two residues from Lys-406 to Phe-404, while the C-terminus of helix 5 is elongated by three residues from Tyr-434 to Ile-437 upon membrane association.
Ho
Derek
D
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
Merrill
A Rod
AR
D000595:Amino Acid Sequence; D001643:Bridged Bicyclo Compounds; D003087:Colicins / Q000737:chemistry* / Q000302:isolation & purification; D003545:Cysteine / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry*; D005454:Fluorescence Polarization; D007473:Ion Channels / Q000737:chemistry* / Q000302:isolation & purification; D008051:Lipid Bilayers / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D050505:Mutant Proteins / Q000737:chemistry; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012996:Solutions; D013050:Spectrometry, Fluorescence; D013194:Staining and Labeling
D001643:Bridged Bicyclo Compounds; D003087:Colicins; D007473:Ion Channels; D008051:Lipid Bilayers; D050505:Mutant Proteins; D012996:Solutions; D003545:Cysteine; C028381:monobromobimane