Locating a residue in the diphtheria toxin channel.
We are studying structure-function relationships in the Diphtheria Toxin (DT) channel using a combination of site-directed mutagenesis and electrophysiology in artificial lipid bilayers. We made site-directed mutations of charged residues in the toxin's channel-forming B fragment, and after expressing the mutant proteins in Escherichia coli, we analyzed the single channels they formed in lipid bilayers. Changing aspartate 352, which is located in a short hydrophilic loop separating two hydrophobic stretches, to asparagine or lysine dramatically reduces the single-channel conductance of the pore at pH 5.3 cis, 7.2 trans (5.3/7.2). Lowering the pH on both sides of the membrane essentially eliminates the difference between wild-type and D352N; this finding is consistent with the idea that an aspartate with a (protonated) neutral side-chain and the always neutral asparagine have similar electrostatic influences on permeant ions. Using a high concentration of permeant buffer to clamp the pH of the cis compartment and the pore, and varying the pH on the trans side, we have located D352 at or near the trans compartment. We further find that D352N channels, in contrast to wild-type, display conductances independent of trans pH. This observation allows us to determine the titration curve of aspartate 352 in the wild-type toxin, establishing its pKa at approximately 5.5.
Mindell
J A
JA
Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461.
Silverman
J A
JA
Collier
R J
RJ
Finkelstein
A
A
D000595:Amino Acid Sequence; D055592:Biophysical Phenomena; D001703:Biophysics; D004167:Diphtheria Toxin / Q000737:chemistry* / Q000235:genetics; D004553:Electric Conductivity; D006863:Hydrogen-Ion Concentration; D007473:Ion Channels / Q000737:chemistry* / Q000235:genetics; D008051:Lipid Bilayers; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013329:Structure-Activity Relationship
D004167:Diphtheria Toxin; D007473:Ion Channels; D008051:Lipid Bilayers