Abstract
Ligand binding to receptors is one of the most important regulatory elements in biology as it is the initiating step in signaling pathways and cascades. Thus, precisely localizing binding sites and measuring interaction forces between cognate receptor–ligand pairs leads to new insights into the molecular recognition involved in these processes. Here we present a detailed protocol about applying a technique, which combines atomic force microscopy (AFM)-based recognition imaging and force spectroscopy for studying the interaction between (membrane) receptors and ligands on the single molecule level. This method allows for the selection of a single receptor molecule reconstituted into a supported lipid membrane at low density, with the subsequent quantification of the receptor–ligand unbinding force. Based on AFM tapping mode, a cantilever tip carrying a ligand molecule is oscillated across a membrane. Topography and recognition images of reconstituted receptors are recorded simultaneously by analyzing the downward and upward parts of the oscillation, respectively. Functional receptor molecules are selected from the recognition image with nanometer resolution before the AFM is switched to the force spectroscopy mode, using positional feedback control. The combined mode allows for dynamic force probing on different pre-selected molecules. This strategy results in higher throughput when compared with force mapping. Applied to two different receptor–ligand pairs, we validated the presented new mode.
Highlights
Atomic force microscopy (AFM) is one of the most versatile microscopic tools due to its capability to provide topographical images, and to probe and characterize interactions on the single molecular level [1]
Despite the fact that peak force tapping (PFT) was successfully applied on a variety of biological samples, ranging from membrane proteins [5] to entire cells [6], for high-resolution maps, a few hours are still required
We describe here a detailed protocol of performing AFM-based force spectroscopy guided by recognition imaging in order to map and characterize binding sites of membrane proteins reconstituted into lipid bilayers
Summary
Atomic force microscopy (AFM) is one of the most versatile microscopic tools due to its capability to provide topographical images, and to probe and characterize interactions on the single molecular level [1]. High densities and homogeneous distributions of receptor molecules are required for performing a successful force spectroscopy experiment, as the tip is “blindly” moved in the z-direction Such conditions are not always applicable, in particular when the molecules of interest are membrane proteins. Low reconstitution efficiencies are often obtained for protein integration into an artificial lipid membrane To overcome this issue, an AFM-technique known as “force volume (FV)” was initially proposed by Cleveland et al [2] and further used in pioneering studies by Heinz and Hoh [3] and Gaub et al [4]. We describe here a detailed protocol of performing AFM-based force spectroscopy guided by recognition imaging in order to map and characterize binding sites of membrane proteins reconstituted into lipid bilayers
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