FhuA interactions in a detergent-free nanodisc environment

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 K-D is similar to 200 nM; 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 (K-D similar to 3.5 nM), 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 beta-barrel membrane proteins in a membrane-like environment. (C) 2013 Elsevier B.V. All rights reserved.