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-l,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.
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