Transmembrane insertion of the colicin Ia hydrophobic hairpin.
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.
Kienker
P K
PK
Department of Physiology & Biophysics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
Qiu
X
X
Slatin
S L
SL
Finkelstein
A
A
Jakes
K S
KS
D001426:Bacterial Proteins; D001710:Biotin; D003087:Colicins / Q000737:chemistry*; D015640:Ion Channel Gating; D007473:Ion Channels / Q000737:chemistry*; D008051:Lipid Bilayers; D011485:Protein Binding; D011487:Protein Conformation; D019809:Streptavidin
D001426:Bacterial Proteins; D003087:Colicins; D007473:Ion Channels; D008051:Lipid Bilayers; D001710:Biotin; D019809:Streptavidin