title: Kinetic intermediate reveals staggered pH-dependent transitions along the membrane insertion pathway of the diphtheria toxin T-domain.
abstract: The pH-triggered membrane insertion pathway of the T-domain of diphtheria toxin was studied using site-selective fluorescence labeling with subsequent application of several spectroscopic techniques (e.g., fluorescence correlation spectroscopy, FRET, lifetime quenching, and kinetic fluorescence). FCS measurements indicate that pH-dependent formation of the membrane-competent form depends only slightly on the amount of anionic lipids in the membrane. The subsequent transbilayer insertion, however, is strongly favored by anionic lipids. Kinetic FRET measurements between the donor-labeled T-domain and acceptor-labeled lipid vesicles demonstrate rapid membrane association at all pH values for which binding occurs. In contrast, the transmembrane insertion kinetics is significantly slower and is also both pH- and lipid-dependent. Analysis of kinetic behavior of binding and insertion indicates the presence of several interfacial intermediates on the insertion pathway of the T-domain, from soluble W-state to transmembrane T-state. Intermediate interfacial I-state can be trapped in membranes with low content of anionic lipids (10%). In membranes of greater anionic lipid content, another pH-dependent transition results in the formation of the insertion-competent state and subsequent transmembrane insertion. Comparison of the results of various kinetic and equilibrium experiments suggests that the pH dependences determining membrane association and transbilayer insertion transitions are different but staggered. Anionic lipids not only assist in formation of the insertion-competent form but also lower the kinetic barrier for the final insertion.
authors: Department of Biochemistry and Molecular Biology, Kansas University Medical Center, Kansas City, Kansas 66160-7421, USA.
authors: Alexander
authors: A
authors: Kyrychenko
authors: Yevgen O
authors: YO
authors: Posokhov
authors: Mykola V
authors: MV
authors: Rodnin
authors: Alexey S
authors: AS
authors: Ladokhin
mesh_terms: D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004167:Diphtheria Toxin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006863:Hydrogen-Ion Concentration; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism*; D008563:Membrane Lipids / Q000737:chemistry / Q000378:metabolism; D008566:Membranes / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
chemical_list: D004167:Diphtheria Toxin; D008051:Lipid Bilayers; D008563:Membrane Lipids; D003545:Cysteine