title: Reversible refolding of the diphtheria toxin T-domain on lipid membranes.
abstract: The catalytic domain of diphtheria toxin (DT) is translocated across endosomal membranes by the T-domain (DTT) in response to acidification. Understanding the energetics of translocation, besides clarifying the mechanism of translocation, should provide insights into general principles of membrane protein stability and assembly. As a first step, we have evaluated the energetics of DTT binding to lipid vesicles using three single-cysteine mutants (L350C, Q369C, and Y280C) labeled with a 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) fluorophore sensitive to polarity changes. Remarkably strong association with the vesicles was detected for all mutants, even at pH 7 at which DTT is believed to be in a fully folded membrane-incompetent state. Lowering the pH in the presence of anionic membranes resulted in a strong but reversible increase in emission of NBD-labeled mutants, consistent with reversible membrane insertion. This reversibility permitted free energies of DTT interactions with vesicles to be determined for the first time. Free energy values for the three mutants ranged from -8 to -10 kcal mol(-1) at pH 4.3 and from -7 to -8 kcal mol(-1) at pH 7. Insights into the disposition of DTT on membranes were obtained using a novel hydropathy analysis that considers the relative free energies of transmembrane and interfacial interactions as a function of pH. This analysis suggests that interactions at the membrane interface dominate pH-triggered insertion of DTT, implying that the folding pathway involves interfacial intermediates.
authors.affiliation: Department of Physiology and Biophysics, University of California, Irvine, California 92697-4560, USA. ladokhin@uci.edu
authors.forename: Alexey S
authors.initials: AS
authors.lastname: Ladokhin
authors.forename: Rachel
authors.initials: R
authors.lastname: Legmann
authors.forename: R John
authors.initials: RJ
authors.lastname: Collier
authors.forename: Stephen H
authors.initials: SH
authors.lastname: White
mesh_terms: D004167:Diphtheria Toxin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D020673:Protein Renaturation; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
chemical_list: D004167:Diphtheria Toxin; D008051:Lipid Bilayers