Effects of Mutations in Proline 345 on Insertion of Diphtheria Toxin into Model Membranes |
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Authors: | H Zhan JL Elliott WH Shen PD Huynh A Finkelstein RJ Collier |
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Institution: | (1) Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA, US;(2) Departments of Physiology & Biophysics and Neuroscience, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA, US |
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Abstract: | Translocation of the catalytic domain of diphtheria toxin (DT) across the endosomal membrane to the cytoplasm of mammalian
cells requires the low-pH-dependent insertion of a hydrophobic helical hairpin (TH8-TH9) that is buried within the T domain
of the native protein. Mutations of Pro345, which terminates helix TH8, have been reported to block toxicity for Vero cells.
We found that mutant toxins in which Pro345 had been replaced by Cys, Glu, or Gly were profoundly defective at low pH in forming
channels in planar phospholipid bilayers and in permeabilizing phospholipid vesicles to entrapped fluorophores. Experiments
with isolated T domain containing a polarity-sensitive fluorophore attached to Cys at position 332 suggest that the P345E
mutation blocks membrane insertion. None of the Pro345 mutations shifted the pH-dependence of binding in solution of the hydrophobic
fluorophore, 2-p-toluidinyl-naphthalene 7-sulfonate. The results indicate that proline at position 345 is required for the T domain to insert
into phospholipid bilayers or to adopt a functional conformation within the bilayer.
Received: 23 July 1998/Revised: 19 October 1998 |
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Keywords: | : Diphtheria toxin — Proline — Mutagenesis — Membrane insertion — Transmembrane domain — Site-specific labeling |
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