Abstract
The membrane-bound closed state of the colicin E1 channel domain was investigated by site-directed fluorescence labeling using a bimane fluorophore attached to each single cysteine residue within helix 2 of each mutant protein. The fluorescence properties of the bimane fluorophore were measured for the membrane-associated form of the closed channel and included fluorescence emission maximum, fluorescence anisotropy, apparent polarity, surface accessibility, and membrane bilayer penetration depth. The fluorescence data show that helix 2 is an amphipathic alpha-helix that is situated parallel to the membrane surface, but it is less deeply embedded within the bilayer interfacial region than is helix 1 in the closed channel. A least squares fit of the various data sets to a harmonic wave function indicated that the periodicity and angular frequency for helix 2 in the membrane-bound state are typical for an amphipathic alpha-helix (3.8 +/- 0.1 residues per turn and 94 +/- 4 degrees, respectively) that is located at an interfacial region of a membrane bilayer. Dual quencher analysis also revealed that helix 2 is peripherally membrane associated, with one face of the helix dipping into the interfacial region of the lipid bilayer and the other face projecting outwardly into the aqueous solvent. Finally, our data show that helices 1 and 2 remain independent helices upon membrane association with a short connector link (Tyr(363)-Gly(364)) and that short amphipathic alpha-helices participate in the formation of a lipid-dependent, toroidal pore for this colicin.
Highlights
100 Ϯ 10 63 Ϯ 17 106 Ϯ 5 81 Ϯ 2 91 Ϯ 9 72 Ϯ 2 86 Ϯ 7 101 Ϯ 11 115 Ϯ 3 108 Ϯ 4 93 Ϯ 3 112 Ϯ 20 75 Ϯ 7 108 Ϯ 9 60 Ϯ 12 a Values shown are the mean Ϯ S.D. of at least triplicate measurements
We showed that helix 1 within the colicin E1 channel domain maintains its amphipathic ␣-helical structure upon binding to the membrane surface in the closed channel state [37] and that the soluble helix consists of residues 350 – 362, whereas the membrane-bound helix consists of residues 347–362 with the Tyr363–Gly364 sequence serving as a helix breaker between helices 1 and 2
Channel activity is insensitive to inactivation by Cys replacement mutagenesis and subsequent bimane labeling [37] (Table 1)
Summary
Unless otherwise stated, were purchased from Sigma. All steady-state fluorescent measurements were collected using a PTI-Alphascan-2 spectrofluorimeter (Photon Technologies Inc, South Brunswick, NJ) equipped with a thermostated cell holder. All measurements are reported as the mean Ϯ S.D. and were performed at least in triplicate. Mutagenesis, Protein Purification, and Monobromobimane Labeling—Cysteine-scanning mutagenesis, in which each amino acid residue from Glu365 to Gly380 of P190H62 (except for K366C) was individually replaced with a cysteine, was performed using the Stratagene (La Jolla, CA) QuikchangeTM mutagenesis kit. Plasmid DNA was purified using the High Pure PlasmidTM isolation kit from Roche Diagnostics. Wild type P190H6, P190H6/C505A (Cys-less wild type), and Cys mutant plasmids were prepared and purified from transformed lexAϪ
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call