NanoBlocks: Creating Genetically Encoded Protein Switches Using Competitive Binding

Many regulatory mechanisms function by changing a molecule's conformation such that it is switched between active and inactive states. Engineering macromolecules such as proteins to show robust conformational changes in response to arbitrary, well-defined molecular stimuli is of great interest in fields such as synthetic biology and nanoscale engineering, because it allows the development of "smart" molecular tools. We present a strategy that can convert "simple" affinity binders such as nanobodies into protein switches that undergo clear structural rearrangements in response to their innate targets, by introducing a peptide that competes for the binding site. We demonstrate proof-of-concept implementations of this "NanoBlock" design by developing genetically encoded fluorescent biosensors based on two entirely different binder frameworks, the ALFA nanobody and the PDZ domain of Erbin. We show that our design allows the construction of well-performing protein switches that can reliably detect their targets in vitro, in mammalian cells, as part of fluorescence-activated cell sorting (FACS) experiments, and that also generalizes to the creation of stimulus-responsive enzymes via the integration of a bioluminescent readout. We furthermore develop a theoretical model that rationalizes the observations and provides insight into the underlying molecular mechanisms. Overall, our strategy expands the possibilities to create "smart" protein-based molecules and effectors.