Fig. 2.

(A) Crystallographic structure of the diphtheria toxin T-domain in soluble form at neutral pH [Bennett & Eisenberg, 1994]. The consensus insertion hairpin consisting of helices TH8 and TH9 is shown solid, while the rest of the structure is shown semitransparent. Two residues, N235 and Q369 (highlighted in CPK), along TH9 were replaced with cysteines (one at a time), providing unique labeling sites for fluorescence dye (NBD or AlexaFluor488) used in steady-state and kinetic measurements of insertion. (B and C) Membrane binding and insertion kinetics of the T-domain measured with LUV of specified lipid compositions (color-coded). (B) Binding kinetics were followed by the change in FRET signal between donor-labeled T-domain and acceptor-labeled LUV. (C) Insertion kinetics were followed by changes in fluorescence intensity of environment-sensitive probe NBD attached to the center region of the membrane-insertion domain (Q369C-NBD). Differences observed in the two types of kinetics reveal the insertion intermediate, which depends on pH and lipid composition. Modified from Kyrychenko, A., Posokhov, Y.O., Rodnin, M.V., Ladokhin, A.S. (2009) Kinetic intermediate reveals staggered pH-dependent transitions along the membrane insertion pathway of the diphtheria toxin T-domain. Biochemistry, 48, 7584–7594.