Transmembrane insertion of the Colicin Ia hydrophobic hairpin

Abstract

Colicin Ia is a bactericidal protein that forms voltage-dependent, ion-conducting channels, both in the inner membrane of target bacteria and in planar bilayer membranes. Its amino acid sequence is rich in charged residues, except for a hydrophobic segment of 40 residues near the carboxyl terminus. In the crystal structure of colicin Ia and related colicins, this segment forms an alpha-helical hairpin. The hydrophobic segment is thought to be involved in the initial association of the colicin with the membrane and in the formation of the channel, but various orientations of the hairpin with respect to the membrane have been proposed. To address this issue, we attached biotin to a residue at the tip of the hydrophobic hairpin, and then probed its location with the biotin-binding protein streptavidin, added to one side or the other of a planar bilayer. Streptavidin added to the same side as the colicin prevented channel opening. Prior addition of streptavidin to the opposite side protected channels from this effect, and also increased the rate of channel opening; it produced these effects even before the first opening of the channels. These results suggest a model of membrane association in which the colicin first binds with the hydrophobic hairpin parallel to the membrane; next the hairpin inserts in a transmembrane orientation; and finally the channel opens. We also used streptavidin binding to obtain a stable population of colicin molecules in the membrane, suitable for the quantitative study of voltage-dependent gating. The effective gating charge thus determined is pH-independent and relatively small, compared with previous results for wild-type colicin Ia.

Calorimetric Investigations of the Structural Stability and Interactions of Colicin B Domains in Aqueous Solution and in the Presence of Phospholipid Bilayers

“…The decreased thermal stability of ColB at lowered pH correlates with the previously observed requirement of acidic conditions for optimal channel and molten globule formation of ColB (30), increased membrane association of ColA (31,32), augmented membrane-insertion rate of ColIa (33,34), and pH-sensitive conformational changes of ColE1 in the presence of membranes (35). The unfolding profile of diphtheria toxin has been published previously (36).…”

Colicin Biology

“…Experiments in planar bilayers (340) showed that the interhelical loop between H8 and H9 of colicin Ia channels is exposed to the solution on the trans side of the membrane in the open state and in at least some closed states (including channels which had never opened), thus demonstrating that the hydrophobic hairpin can adopt a transmembrane orientation in the closed state, before the channel opens.…”

Structural studies of apoptosis and ion transport regulatory proteins in membranes

“…48 We find that this structural similarity between the soluble forms of the two proteins appears to extend to their membrane associated structures. For colicin, insertion of the hydrophobic helical hairpin into the membrane is supported by planar bilayer experiments, 49 fluorescence energy transfer 50 and solid-state NMR. 51,52 Membrane association occurs when the colicin soluble helical bundle unfolds, exposing the hydrophobic helical hairpin which inserts in the membrane, while the rest of the protein forms a helical network on the membrane surface, adopting a so-called umbrella conformation.…”