Linked Life Data

8125966

Source:http://linkedlifedata.com/resource/pubmed/id/8125966

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
10
pubmed:dateCreated
1994-4-12
pubmed:abstractText
Channel-forming colicins are Escherichia coli proteins that form voltage-dependent channels in lipid bilayer membranes and are lethal to sensitive strains of E. coli. Experiments with colicin E1 have led to a model of voltage dependence based on the insertion of alpha-helical segments of the protein into the membrane in response to cis-positive voltages. This model was tested on the partly homologous colicin Ia protein, which offers certain advantages over colicin E1 as a model channel, it is active at neutral pH and exhibits comparatively well-defined single channel conductance. We describe here the creation of a specific probe for locating a particular amino acid residue on one side or the other of a planar lipid bilayer membrane, by using the biotin-streptavidin system. Site-directed mutagenesis was used to change lysine 544 of colicin Ia to cysteine. This placed a unique cysteine at a site expected, by homology to colicin E1, to cross the membrane from the cis to the trans side in association with the opening of the channel. This unique cysteine was biotinylated chemically, so that it could serve as a target for streptavidin. Incubation of the biotinylated mutant colicin with streptavidin blocked its killing activity, in vivo; incubation of wild-type colicin, which lacks cysteine, with streptavidin, did not affect its activity. Channels formed by the biotinylated mutant protein in planar lipid bilayers were abolished by streptavidin added to the cis side of the membrane, if the channels were closed, but not if they were open. Trans streptavidin had no effect on either open or closed channels. Thus, when the channel is closed, residue 544 of colicin Ia is accessible to cis streptavidin in the closed state, but the opening of the channel eliminates this accessibility.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Mar
pubmed:issn
0021-9258
pubmed:author
pubmed:issnType
Print
pubmed:day
11
pubmed:volume
269
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
7483-8
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
1994
pubmed:articleTitle
Site-specific biotinylation of colicin Ia. A probe for protein conformation in the membrane.
pubmed:affiliation
Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S.