Locating a residue in the diphtheria toxin channel

J. A. Mindell, J. A. Silverman, R. J. Collier, A. Finkelstein

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

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 pK(a) at ~5.5.

Original languageEnglish (US)
Pages (from-to)41-44
Number of pages4
JournalBiophysical Journal
Volume62
Issue number1
StatePublished - 1992

Fingerprint

Diphtheria Toxin
Aspartic Acid
Asparagine
Lipid Bilayers
Electrophysiology
Mutant Proteins
Site-Directed Mutagenesis
Static Electricity
Lysine
Buffers
Ions
Escherichia coli
Mutation
Membranes

ASJC Scopus subject areas

  • Biophysics

Cite this

Mindell, J. A., Silverman, J. A., Collier, R. J., & Finkelstein, A. (1992). Locating a residue in the diphtheria toxin channel. Biophysical Journal, 62(1), 41-44.

Locating a residue in the diphtheria toxin channel. / Mindell, J. A.; Silverman, J. A.; Collier, R. J.; Finkelstein, A.

In: Biophysical Journal, Vol. 62, No. 1, 1992, p. 41-44.

Research output: Contribution to journalArticle

Mindell, JA, Silverman, JA, Collier, RJ & Finkelstein, A 1992, 'Locating a residue in the diphtheria toxin channel', Biophysical Journal, vol. 62, no. 1, pp. 41-44.
Mindell JA, Silverman JA, Collier RJ, Finkelstein A. Locating a residue in the diphtheria toxin channel. Biophysical Journal. 1992;62(1):41-44.
Mindell, J. A. ; Silverman, J. A. ; Collier, R. J. ; Finkelstein, A. / Locating a residue in the diphtheria toxin channel. In: Biophysical Journal. 1992 ; Vol. 62, No. 1. pp. 41-44.
@article{d7dda4995f0840e88ea96bf38e0ae42a,
title = "Locating a residue in the diphtheria toxin channel",
abstract = "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 pK(a) at ~5.5.",
author = "Mindell, {J. A.} and Silverman, {J. A.} and Collier, {R. J.} and A. Finkelstein",
year = "1992",
language = "English (US)",
volume = "62",
pages = "41--44",
journal = "Biophysical Journal",
issn = "0006-3495",
publisher = "Biophysical Society",
number = "1",

}

TY - JOUR

T1 - Locating a residue in the diphtheria toxin channel

AU - Mindell, J. A.

AU - Silverman, J. A.

AU - Collier, R. J.

AU - Finkelstein, A.

PY - 1992

Y1 - 1992

N2 - 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 pK(a) at ~5.5.

AB - 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 pK(a) at ~5.5.

UR - http://www.scopus.com/inward/record.url?scp=0026643615&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0026643615&partnerID=8YFLogxK

M3 - Article

VL - 62

SP - 41

EP - 44

JO - Biophysical Journal

JF - Biophysical Journal

SN - 0006-3495

IS - 1

ER -