ABSTRACTDNA origami nanostructures provide a unique platform for the direct observation of protein-protein interactions at the single-molecule level. Here, we used DNA origami in combination with fast-scan atomic force microscopy to observe the activation-induced dissociation of individual protein kinase A (PKA) holoenzymes. The PKA holoenzyme consists of two regulatory (R) and two catalytic (C) subunits. When cAMP binds to the R subunits it causes dissociation of the C subunits from the R subunit dimer and activation of the enzyme. Using a DNA origami platform, we were able to observe the activation of PKA in response to photolysis of caged cAMP. Furthermore, exploiting the potential of DNA origami for precise positioning of biomolecules, we were able to position the catalytic subunit of adenylyl cyclase in close proximity to PKA and to observe the activation of PKA in response to cAMP produced by adenylyl cyclase. We provide tools for the observation of signalling pathways at the single-molecule level and show that the C subunits of PKA dissociate from the holoenzyme but stay within ~10 nm of the R subunit dimer upon activation.
Interaction of DNA-dependent protein kinase with DNA and with Ku: biochemical and atomic-force microscopy studies