Combined Single Molecule Recognition Imaging and Force Spectroscopy to Study the Interactions Between Uncoupling Proteins and Purine Nucleotides

Abstract

We combined recognition imaging and force spectroscopy so as to study the interactions between receptors and ligands on the single molecule level. This method allowed the selection of a single receptor molecule reconstituted in a supported lipid membrane at low density, with the subsequent quantification of the receptor-ligand unbinding force. Based on atomic force microscopy (AFM) tapping mode a cantilever tip carrying a ligand molecule was oscillated across a membrane. Topography and recognition Images of reconstituted receptors were recorded simultaneously by analysing the downwards and upwards parts of the oscillation, respectively. Functional receptor molecules were selected from the recognition image with nanometer resolution before the AFM was switched to the force spectroscopy mode, using positional feedback control. The combined mode allowed for dynamic force probing on different pre-selected molecules, resulting in a higher throughput when compared with force mapping. We applied this method for a quantitative characterization of the interaction between uncoupling proteins (UCP) and purine nucleotides (PN). The UCPs are mitochondrial proton transporters, which are proposed to be involved in thermogenesis, reactive oxygen sites (ROS) regulation and metabolism. Recently we hypothesized that PNs bind to UCP1 from cis- and transsite, although only cis-binding led to protein inhibition [1]. So as to get better insight into the molecular mechanism of this interaction we characterized the oligomeric state of UCPs reconstituted in lipid membranes and probed their interaction force with PNs using dynamic force spectroscopy. In the latter mode, the dynamics of force loading was varied, which elucidated the recognition dynamics and yielded information about binding pocket, binding energy barriers, and chemical reaction rates. This study was supported by the FWF project FWFP25357000. [1] Zhu, et al. JACS 135 (2013) 3640.