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

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 ␣-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 ␣-helix (3.8 ؎ 0.1 residues per turn and 94 ؎ 4°, 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 ␣-helices participate in the formation of a lipiddependent, toroidal pore for this colicin.

Resolving the 3D spatial orientation of helix I in the closed state of the colicin E1 channel domain by FRET. Insights into the integration mechanism

“…In particular, the "compactness" of the new topology allows interaction of H 1 with H 8/9 that might serve to stabilize the hairpin configuration of H 1 and H 2 . The same FRET distance between 355DABMI (at H 1 ) and 369COU (at H 2 ) in both the soluble and the membrane-bound proteins [33], and the same pattern of mobility of the segment 355e363 (the tip H 1/2 hairpin) between the soluble and the membrane-bound states [18,19], support the preservation of the H 1/2 super-secondary structure. Besides, the location of the H 1/2 hairpin the new model is also compatible with a comprehensive mechanism of insertion and unfolding on the membrane (see below).…”

Computational studies of colicin insertion into membranes: The closed state

“…Solid‐state NMR experiments also support this model for colicin Ia 39, 51. Site directed fluorescence labeling shows that the amphipathic helices are spread on the membrane surface 34, 52–55. However, differential scanning calorimetry (DSC) experiments suggest that the tertiary structure is retained at least to some degree in the inserted state 56…”

“…For the membrane‐inserted state a variety of experimental data are available, though of low resolution. There is evidence from site‐directed fluorescence labeling experiments that the amphipathic helices in colicin E1 are adsorbed to the membrane surface to a higher or lower degree 34, 52. Quenching experiments using brominated lipids on colicin A have shown that the tryptophans in this colicin penetrate the membrane, also pointing to a membrane surface location of the amphipathic helices 72.…”

Harmonic Analysis of the Fluorescence Response of Bimane Adducts of Colicin E1 at Helices 6, 7, and 10

“…To resolve the issue, an additional eight variants were prepared using the C505A mutation to facilitate the preparation of single-Cys variants. It was previously reported that the C505A mutation does not perturb the folded structure and does not impair channel activity (30). Labeling efficiency was significantly restored with the incorporation of C505A mutation with the above approach except for the I454C variant (Fig.…”

“…The ratio of quenching by 10-DN relative to that by KI (Q-ratio) was used to determine the penetration depth of bimane in lipid bilayers as described previously (30). The Q-ratio was calculated from the formula,…”