Thermodynamics of Interfacial Membrane Binding and Transmembrane Insertion of Diphtheria Toxin T-Domain: Fluorescence Correlation Spectroscopy Study

Abstract

Recent studies of kinetic behavior of binding and insertion of diphtheria toxin translocation domain (DTT) into lipid membranes [Kyrychenko et al. Biochemistry 2009, 48:7584] revealed the presence of several interfacial intermediates on the insertion pathway leading from soluble W-state to transmembrane T-state. It has been found that an intermediate interfacial I-state can be trapped in membranes with low content of anionic lipids (10%), while in membranes of greater anionic lipid content, another pH-dependent transition results in the formation of the insertion-competent state and subsequent transmembrane insertion. In this work we applied fluorescence correlation spectroscopy (FCS) to determine the free energy (ΔG) stabilizing final transmembrane and interfacial intermediate states. To avoid aggregation of DTT and to chaperone its membrane insertion, the FCS measurements were performed in the presence of fluorinated surfactants FTAC-C6. Our results indicate that the free energy of binding (ΔG) to lipid vesicles with formation of trapped interfacial intermediate state is about −8 kcal/mole, and this ΔG value does not change with pH, while the ΔG difference between transmembrane state and the interfacial state ranges from −1.5÷-4 kcal/mole depending on membrane lipid composition and pH of media. Our results confirm the interface-directed model of spontaneous insertion of non-constitutive membrane proteins and provide an important benchmark for future measurements of ΔG stabilizing the structure of constitutive membrane proteins. Supported by NIH GM069783(-04S1)