Thermodynamic and Kinetic Analysis of Membrane Protein Insertion: Case of Diphtheria Toxin T‐Domain

Abstract

The pH‐triggered membrane insertion pathway of the diphtheria toxin T‐domain was studied using site‐selective fluorescence labeling with subsequent application of several spectroscopic techniques. FRET‐based kinetic binding measurements demonstrate rapid association which is largely lipid‐independent. In contrast, the transmembrane insertion kinetics is significantly slower, and is also both pH‐ and lipid‐dependent. Kinetic analysis 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. Fluorescence correlation spectroscopy measurements of surfactant‐chaperoned membrane insertion indicate that the free energies of stabilizing the interfacial intermediate and the final transmembrane states are −8 and −12 kcal/mole, respectively. We conclude that the acid‐induced membrane action of the T‐domain is modulated by the following two titration transitions with the staggered pH‐dependencies: (a) formation of the membrane‐competent state in bulk solution and (b) formation of the insertion‐competent state on the membrane interface. Anionic lipids not only assist in formation of the insertion‐competent form, but also lower the kinetic barrier for the final insertion. NIH GM069783.