The goal of the proposed research is to generate genetically encoded bioluminescent tags for live-cell luminescence-based photoactivated localization microscopy (L-PALM). This revolutionary mode of superresolution imaging will maintain all of the benefits of fluorescence PALM (fPALM) but will eliminate the need for excitation light. fPALM is largely unsuitable for imaging live cells because it requires high excitation intensities that lead to phototoxicity. Because luminescence generates light without the need for external excitation, L-PALM will not suffer from this limitation. The latest generation of genetically encoded bioluminescent labels are well suited for widefield microscopy of subcellular structures, but are still approximately 1000-fold too dim to be used for single-molecule localization on practical time scales. To remedy this deficiency, this study is designed to produce bioluminescent probes with photon output rates sufficient to localize ~100,000 molecules in one minute. To generate this increased output, luciferases will first be coupled to our brightest fluorescent proteins to maximize luminescence quantum yield via the Förster resonance energy transfer mechanism. Once maximal output is achieved in this first step, the luciferase portion of the fusion will then be subjected to structure-guided directed evolution targeted at lowering oxyluciferin binding affinity and thus increasing the catalytic rate of the enzyme. Such alterations are predicted to reduce the luminescence quantum yield of the luciferase, but energy transfer to a fluorescent protein will rescue the luminescence, allowing much faster enzymes to be engineered with this strategy. To be useful for live-cell L-PALM, bioluminescent probes must also be capable of switching on and off controllably to prevent signal overlap between individual molecules in each image frame. Two independent mechanisms for producing switchable light output will be pursued in this project: (1) optimization of energy transfer between luciferases and photoswitchable fluorescent proteins, followed by directed evolution to increase light output and improve switching kinetics;; (2) insertion of light-modulated domains into split luciferases in order to allosterically control enzyme activity. Throughout the project, heavy emphasis will be placed on Rosetta-based structure-guided computational design for generating novel luciferase-fluorescent protein fusion topologies, altering luciferase active site environments, and engineering allosterically-regulated luciferases. Directed evolution with image-based screening will then be the primary approach for improving the properties of probes under development in each aim. The end products of this project will be a set of genetically encoded bioluminescent probes with brightness and photoswitching properties suitable for the development of L- PALM methodologies. Beyond their ultimate utility for L-PALM imaging, man...