ABSTRACT Among the environmental factors that influence biology, light controls an incredibly rich set of processes including circadian rhythms, movement and development. Central to this regulation are diverse families of photoreceptors that harness the photochemically-triggered configurational changes of light-sensitive cofactors, the chemical properties of which confer sensitivity to specific portions of the UV and visible spectrum. These structural alterations are amplified as allosteric changes in the surrounding protein, initiating signal transduction pathways that control biological responses. Understanding how these initial sensing and signaling events are triggered has given insight into fundamental aspects of biology and enabled the development of novel optogenetic tools that are enabling discoveries in diverse fields of biomedicine. Here we focus on examining both signaling mechanism and applicability to tool development for proteins containing LOV (Light-Oxygen- Voltage) photosensory domains. These domains are found in thousands of proteins to date, controlling the activity of over 20 enzymatic and non-enzymatic effector domains in natural systems and many others in engineered proteins. To do so, LOV domains undergo the specific photochemical formation of protein/flavin adducts upon illumination with blue light, using this as a trigger to allosterically drive protein conformational changes around the flavin. This configurational change is allosterically transmitted to the surrounding protein, providing a light-based “switch” of protein activity that remains engaged until illumination ceases. Fundamental questions regarding these signaling processes remain unanswered, limiting our understanding of natural and engineered LOV proteins. We propose to answer these limitations by pursuing three aims: 1). Determine the lit state structures of two classes of activated LOV proteins to give insight into signaling and guiding future engineering efforts; 2). Establish the correlation of in vitro photochemical and DNA-binding parameters on cellular function of a LOV-controlled transcription factor tools; 3). Examine the generality of LOV signaling models by determining the structural role of photosensing in a novel class of RGS-LOV signaling proteins. To achieve these ends, we will take advantage of an broad foundation of preliminary structural and functional data that will be extended with a mix of biophysical and biochemical studies. Outcomes from this research will include information about fundamental regulatory processes employed by these proteins, giving insights that will be broadly applicable for signal transduction studies.