TonB-dependent membrane receptors from bacteria have been analyzed in detergent-containing solution, an environment that may influence the role of ligand in inducing downstream interactions. We report reconstitution of FhuA into a membrane mimetic: nanodiscs. In contrast to previous results in detergent, we show that binding of TonB to FhuA in nanodiscs depends strongly on ferricrocin. The stoichiometry of interaction is 1:1 and the binding constant KD is ~200nM; an equilibrium affinity that is ten-fold lower than reported in detergent. FhuA in nanodiscs also forms a high-affinity binding site for colicin M (KD ~3.5nM), while ferricrocin renders FhuA refractory to colicin binding. Together, these results demonstrate the importance of the ligand in regulating receptor interactions and the advantages of nanodiscs to study β-barrel membrane proteins in a membrane-like environment.
Keywords: BN; CN; Channels; DDM; Fc; ITC; LDAO; MSP; Membrane; N-lauryldimethyl amine-N-oxide; Nanodiscs; Siderophore; Transporters; blue-native; clear-native; dodecyl-β-d-maltoside; ferricrocin; isothermal titration calorimetry; membrane scaffold protein; β-Barrel proteins.
© 2013.