Scanning the Membrane-bound Conformation of Helix 1 in the Colicin E1 Channel Domain by Site-directed Fluorescence Labeling

Abstract

Helix 1 of the membrane-associated closed state of the colicin E1 channel domain was studied by site-directed fluorescence labeling where bimane was covalently attached to a single cysteine residue in each mutant protein. A number of fluorescence properties of the tethered bimane fluorophore were measured in the membranebound state of the channel domain, including fluorescence emission maximum, fluorescence quantum yield, fluorescence anisotropy, membrane bilayer penetration depth, surface accessibility, and apparent polarity. The data show that helix 1 is an amphipathic ␣-helix that is situated parallel to the membrane surface. A least squares fit of the various data sets to a harmonic function indicated that the periodicity and angular frequency for helix 1 are typical for an amphipathic ␣-helix (3.7 ؎ 0.1 residues per turn and 97 ؎ 3.0°, respectively) that is partially bathing into the membrane bilayer. Dual fluorescence quencher analysis also revealed that helix 1 is peripherally membrane-associated, with one face of the helix dipping into the lipid bilayer and the other face projecting toward the solvent. Finally, our data suggest that the helical boundaries of helix 1, at least at the C-terminal region, remain unaffected upon binding to the surface of the membrane in support of a toroidal pore model for this colicin.The colicins are a family of antimicrobial proteins that are secreted by Escherichia coli strains under environmental stress, because of nutrient depletion or overcrowding, and these proteins often target sensitive bacterial strains (1). The lethal actions of colicins against their target cells are manifested in a number of different modes that include the following: (i) formation of depolarizing ion channels in the cytoplasmic membrane, (ii) inhibition of protein and peptidoglycan synthesis, and (iii) degradation of cellular nucleic acids (1-7). In this context, the bacterial machinery responsible for colicin biological activity feature important mechanisms that are fundamental to various biological processes. These mechanisms include protein receptor binding, membrane translocation, membrane binding and protein unfolding, membrane insertion, voltage-gated ion channel formation, catalysis, and inhibition of enzymes.Colicin E1 is a member of the channel-forming subfamily of colicins and is secreted by E. coli that harbors the naturally occurring colE1 plasmid; the whole colicin consists of three functional segments, the translocation, receptor-binding domains, and channel-forming domains. Initially, the receptor-binding domain (8) interacts with the vitamin B 12 receptor of target cells (9). Following receptor recognition, the translocation domain associates with the tolA gene product, which permits the translocation of colicin E1 across the outer membrane and into the periplasm (10). In the periplasm, the channel domain undergoes a conformational change to an insertion-competent state and then inserts spontaneously into the cytoplasmic membrane of the host cell, forming an ion channel. The cha...

Lipid Dependence of the Channel Properties of a Colicin E1-Lipid Toroidal Pore

“…Previously (43,44), we proposed the applicability of a toroidal model for the mechanism of the colicin E1 pore formation, which was recently supported by the data of Musse et al (93). Presently, this model is supported by four additional lines of evidence: (i) the ability of P178 to induce lipid flip-flop (Fig.…”

Toward Elucidating the Membrane Topology of Helix Two of the Colicin E1 Channel Domain

“…Steady-state Intrinsic Trp Fluorescence-The folding properties of all proteins were examined using intrinsic Trp fluorescence as described previously (37). Briefly, WT P190H 6 , P190H 6 /C505A, and unlabeled and bimane-labeled mutant proteins were diluted to 4 M in PBS (50 mM NaH 2 PO 4 , 50 mM Na 2 HPO 4 , 100 mM NaCl, pH 7.0).…”

“…Plasmid DNA was purified using the High Pure Plasmid TM isolation kit from Roche Diagnostics. Wild type P190H 6 , P190H 6 /C505A (Cys-less wild type), and Cys mutant plasmids were prepared and purified from transformed lexA Ϫ E. coli IT3661 cells as described previously (37). The purity of each protein was assessed by SDS-PAGE.…”

“…Asolectin (Fluka, Oakville, Ontario, Canada) was purified according to Schendel and Reid (38), and vesicles were prepared as described earlier (39). Phospholipid concentration was determined using the micro-Bartlett assay (37).…”

“…Purified mutant proteins were labeled with monobromobimane (mBBr) (Molecular Probes, Eugene, OR) at a 20:1 molar ratio (probe:protein), and labeling efficiency was determined as described previously (37). Preparation of Large Unilamellar Vesicles (LUVs)-1,2-Dioleoyl-sn-glycero-3-phosphocholine:1,2-dioleoyl-sn-glycero-3-[phospho-(1-glycerol)] vesicles (60:40% molar ratio) (Avanti Polar Lipids, Alabaster, AL) were prepared and quantified as described previously (37), except the buffer used to resuspend vesicles was 10 mM DMG, 100 mM NaCl, pH 4.0. Asolectin (Fluka, Oakville, Ontario, Canada) was purified according to Schendel and Reid (38), and vesicles were prepared as described earlier (39).…”