Single molecule binding dynamics measured with atomic force microscopy

We present a new method to analyse simultaneous Topography and RECognition Atomic Force Microscopy data such that it becomes possible to measure single molecule binding rates of surface bound proteins. We have validated this method on a model system comprising a S-layer surface modified with Strep-tagII for binding sites and strep-tactin bound to an Atomic Force Microscope tip through a flexible Poly-Ethylene-Glycol linker. At larger distances, the binding rate is limited by the linker, which limits the diffusion of the strep-tactin molecule, but at lateral distances below 3nm, the binding rate is solely determined by the intrinsic molecular characteristics and the surface geometry and chemistry of the system. In this regime, K_on as determined from single molecule TREC data is in agreement with K_on determined using traditional biochemical methods.