Identification of a translocated protein segment in a voltage-dependent channel.
Voltage-gated channels undergo a conformational change in response to changes in transmembrane voltage. Here we use site-directed biotinylation to create conformation-sensitive sites on colicin Ia, a bacteriocidal protein that forms a voltage-sensitive membrane channel, which can be monitored by electrophysiological methods. We investigated a model of gating developed for the partly homologous colicin E1 that is based on the insertion of regions of the protein into the membrane in response to cis-positive voltages. Site-directed cysteine mutagenesis, followed by chemical modification, was used to attach a biotin molecule covalently to a series of unique sites on colicin Ia. The modified protein was incorporated into planar lipid membranes, where the introduced biotin moiety served as a site to bind the water-soluble protein streptavidin, added to one side of the membrane or the other. Our results show that colicin gating is associated with the translocation across the membrane of a segment of the protein of at least 31 amino acids.
Slatin
S L
SL
Albert Einstein College of Medicine, Department of Physiology and Biophysics, Bronx, New York 10461.
Qiu
X Q
XQ
Jakes
K S
KS
Finkelstein
A
A
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000494:pharmacology; D001692:Biological Transport; D001710:Biotin; D003087:Colicins / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004594:Electrophysiology; D015640:Ion Channel Gating; D007473:Ion Channels / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000378:metabolism*; D011487:Protein Conformation; D019809:Streptavidin
D001426:Bacterial Proteins; D003087:Colicins; D007473:Ion Channels; D010446:Peptide Fragments; D001710:Biotin; D019809:Streptavidin; D003545:Cysteine