Probing the structure of the diphtheria toxin channel

Reactivity in planar lipid bilayer membranes of cysteine-substituted mutant channels with methanethiosulfonate derivatives

Paul D. Huynh, Can Cui, Hangjun Zhan, Kyoung Joon Oh, R. John Collier, Alan Finkelstein

Research output: Contribution to journalArticle

44 Citations (Scopus)

Abstract

Previous work has established that the 61 amino acid stretch from residue 322 to 382 in the T-domain of diphtheria toxin forms channels indistinguishable in ion-conducting properties from those formed by the entire T-domain. In the crystal structure of the toxin's water-soluble form, the bulk of this stretch is an α-helical hair-pin, designated TH8-9. The present study was directed at determining which residues in TH8-9 line the ion-conducting pathway of the channel; i.e., its lumen or entrances. To this end, we singly mutated 49 of TH8-9's 51 residues (328-376) to cysteines, formed channels with the mutant T-domain proteins in planar lipid bilayers, and then determined whether they reacted with small, charged, lipid- insoluble, sulfhydryl-specific methanethiosulfonate (MTS) derivatives added to the bathing solutions. The indication of a reaction, and that the residue lined the ion-conducting pathway, was a sudden change in single-channel conductance and/or flickering behavior. The results of this study were surprising in two respects. First, of the 49 cysteine-substituted residues in TH8-9 tested, 23 reacted with MTS derivatives in a most unusual pattern consisting of two segments: one extending from 329 to 341 (11 of 13 reacted), and the other from 347 to 359 (12 of 13 reacted); none of the residues outside of these two segments appeared to react. Second, in every cysteine mutant channel manifesting an MTS effect, only one transition in single- channel conductance (or flickering behavior) occurred, not the several expected for a multimeric channel. Our results are not consistent with an α- helical or β-strand model for the channel, but instead suggest an open, flexible structure. Moreover, contrary to common sense, they indicate that the channel is not multimeric but is formed from only one TH8-9 unit of the T-domain.

Original languageEnglish (US)
Pages (from-to)229-242
Number of pages14
JournalJournal of General Physiology
Volume110
Issue number3
DOIs
StatePublished - Sep 1997

Fingerprint

Diphtheria Toxin
Lipid Bilayers
Cysteine
Ions
Membranes
Lipids
Amino Acids
Water
methanethiosulfonate

Keywords

  • Channel conductance
  • Channel flickering
  • Helical hairpin
  • Streptavidin
  • Subunits

ASJC Scopus subject areas

  • Physiology

Cite this

Probing the structure of the diphtheria toxin channel : Reactivity in planar lipid bilayer membranes of cysteine-substituted mutant channels with methanethiosulfonate derivatives. / Huynh, Paul D.; Cui, Can; Zhan, Hangjun; Oh, Kyoung Joon; Collier, R. John; Finkelstein, Alan.

In: Journal of General Physiology, Vol. 110, No. 3, 09.1997, p. 229-242.

Research output: Contribution to journalArticle

Huynh, Paul D. ; Cui, Can ; Zhan, Hangjun ; Oh, Kyoung Joon ; Collier, R. John ; Finkelstein, Alan. / Probing the structure of the diphtheria toxin channel : Reactivity in planar lipid bilayer membranes of cysteine-substituted mutant channels with methanethiosulfonate derivatives. In: Journal of General Physiology. 1997 ; Vol. 110, No. 3. pp. 229-242.
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abstract = "Previous work has established that the 61 amino acid stretch from residue 322 to 382 in the T-domain of diphtheria toxin forms channels indistinguishable in ion-conducting properties from those formed by the entire T-domain. In the crystal structure of the toxin's water-soluble form, the bulk of this stretch is an α-helical hair-pin, designated TH8-9. The present study was directed at determining which residues in TH8-9 line the ion-conducting pathway of the channel; i.e., its lumen or entrances. To this end, we singly mutated 49 of TH8-9's 51 residues (328-376) to cysteines, formed channels with the mutant T-domain proteins in planar lipid bilayers, and then determined whether they reacted with small, charged, lipid- insoluble, sulfhydryl-specific methanethiosulfonate (MTS) derivatives added to the bathing solutions. The indication of a reaction, and that the residue lined the ion-conducting pathway, was a sudden change in single-channel conductance and/or flickering behavior. The results of this study were surprising in two respects. First, of the 49 cysteine-substituted residues in TH8-9 tested, 23 reacted with MTS derivatives in a most unusual pattern consisting of two segments: one extending from 329 to 341 (11 of 13 reacted), and the other from 347 to 359 (12 of 13 reacted); none of the residues outside of these two segments appeared to react. Second, in every cysteine mutant channel manifesting an MTS effect, only one transition in single- channel conductance (or flickering behavior) occurred, not the several expected for a multimeric channel. Our results are not consistent with an α- helical or β-strand model for the channel, but instead suggest an open, flexible structure. Moreover, contrary to common sense, they indicate that the channel is not multimeric but is formed from only one TH8-9 unit of the T-domain.",
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AU - Collier, R. John

AU - Finkelstein, Alan

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