Author's personal copy Examining the Dynamic Energy Landscape of an Antiporter upon Inhibitor Binding Alexej Kedrov 1 , Matthias Appel 2 , Hella Baumann 1 , Christine Ziegler 2 and Daniel J. Muller 1 ⁎ 1 Biotechnology Center, University of Technology, 01307 Dresden, Germany 2 Max-Planck-Institute of Biophysics, 60438 Frankfurt/Main, Germany Received 27 July 2007; received in revised form 8 November 2007; accepted 12 November 2007 Available online 19 November 2007 Previously, we applied single-molecule force spectroscopy to detect and locate interactions within the functional Na + /H + antiporter NhaA from Escherichia coli. It was observed that the binding of the inhibitor 2- aminoperimidine established interactions different from those introduced by the binding of the native ligand. To understand the inhibitory mechanism of the inhibitor, we applied single-molecule dynamic force spectroscopy to reconstruct the energy landscape of NhaA. Dynamic force spectroscopy revealed that the energy landscape of the antiporter remained mainly unchanged except for the energy barrier of the functionally important transmembrane α-helix IX. Inhibitor binding set this domain into a newly formed deep and narrow energy minimum that kinetically stabilized α-helix IX and reduced its conformational entropy. The entropy reduction of α-helix IX is thought to inhibit its functionally important structural flexibility, while the deeper energy barrier shifted the population of active antiporters towards inhibited antiporters. © 2007 Elsevier Ltd. All rights reserved. Edited by W. Baumeister Keywords: atomic force microscopy; SMFS; single-molecule force spectro- scopy; NhaA; Na + /H + antiporter of Escherichia coli Introduction Intermolecular binding and reactions regulate biological processes. Binding of a messenger mole- cule to a receptor initiates a cascade of cellular responses, interactions between membrane proteins modulate their function, and docking of an inhibitor regulates the protein activity. 1,2 The low energy requirements for switching these non- covalent interactions ensure fast biological pro- cesses. In the last decade, considerable progress has been made in combining experimental data with theoretical considerations to understand the mechanisms behind the switching of functional states on structural, thermodynamic, and kinetic levels. Introducing molecular interactions into the framework of energy landscapes, such as those commonly used to describe protein folding, 3 suggested a convenient and robust approach to interpret biological processes. 4,5 Accordingly, inter- actions such as those introduced by the binding of a small molecule contribute to the conformational energy of a protein and alter its conformational space. This suggests that the protein energy land- scape is highly dynamic, with the positions of energy valleys and barriers reflecting its functional state. Introduced to probe interactions between ligand and receptor, 6,7 single-molecule force spectroscopy (SMFS) has been extended to characterize antibody– antigen recognition 8 as well as the unfolding and refolding of water-soluble proteins 9 and to probe the adhesion of cellular membranes at molecular resolution. 10,11 SMFS with an atomic force micro- scope (AFM) involves tethering a single protein or several proteins between a support and an AFM stylus and applying a mechanical pulling force (Fig. 1a). 12,13 Recording the force over the pulling distance yields a force–distance (F–D) curve in which single force peaks reflect the rupture of intra- and inter- *Corresponding author. E-mail address: mueller@biotec.tu-dresden.de. Abbreviations used: AF, atomic force; AP, 2-aminoperimidine; DFS, dynamic force spectroscopy; F–D, force–distance; NhaA, Na + /H + antiporter of Escherichia coli; SMFS, single-molecule force spectroscopy; bFN , average unfolding force; k u , equilibrium unfolding rate; lr, force-loading rate; x u , distance between the folded state and the transition state. doi:10.1016/j.jmb.2007.11.032 J. Mol. Biol. (2008) 375, 1258–1266 Available online at www.sciencedirect.com 0022-2836/$ - see front matter © 2007 Elsevier Ltd. All rights reserved.