Tilted, Extended, and Lying in Wait:  The Membrane-Bound Topology of Residues Lys-381−Ser-405 of the Colicin E1 Channel Domain

• Published in: Biochemistry (Easton) 2007-05, Vol.46 (20), p.6074-6085
• Main Authors: Wei, Zhikui, White, Dawn, Wang, Jie, Musse, Abdiwahab A, Merrill, A. Rod
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Colicins - chemistry; Colicins - genetics; Colicins - metabolism; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Lipid Bilayers - chemistry; Lipid Bilayers - metabolism; Lysine - genetics; Membrane Potentials; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Peptide Fragments - chemistry; Peptide Fragments - genetics; Peptide Fragments - metabolism; Protein Binding - genetics; Protein Structure, Secondary - genetics; Protein Structure, Tertiary - genetics; Serine - genetics
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi700317k

The membrane-bound closed state (zero potential) of the helix 3 segment (Lys-381−Ser-405) of the colicin E1 channel domain was investigated by site-directed fluorescence labeling using a bimane probe tethered to a single cysteine residue of each mutant protein. A number of fluorescence properties of the tethered bimane probe were measured for the soluble channel mutant proteins as well as for the membrane-bound proteins. A new method called helical periodicity surface analysis was employed to fit the fluorescence data to a harmonic wave function using four different statistical methods. The fit of the various data sets to a harmonic wave function indicated that the periodicity of helix 3 in the membrane-bound state is typical for an amphipathic α helix (3.7−4.0 residues per turn and an angular frequency between 90 and 97°). Notably, upon membrane binding, helix 3 elongates from 15 residues (soluble structure) to 20 residues by a three- and two-residue extension at the N- and C-termini of the helix, respectively. Dual quencher analysis also revealed that helix 3 is appressed to the surface of the membrane with its N-terminus more deeply buried within the interfacial region of the bilayer than its C-terminus. Finally, contrary to a previous report, our data show that helices 3 and 4 remain separate and independent helices upon membrane association in the absence of a membrane potential.