Abstract 1496 Hybrid Fluorescent Protein and SNAP-tag Biosensors for Extracellular ATP

The significance of adenosine 5'-triphosphate (ATP) extends beyond its role as a ubiquitous "energy currency" for all living organisms. It also plays a crucial role in purinergic signaling, a type of cell to cell communication that is mediated by extracellular molecules such as ATP. Purinergic signaling is involved in various fundamental physiological processes including normal neuron-glia communication. It is also associated with pathologies of traumatic brain injuries and inflammation. Our broad goal is to develop reliable and accurate optical tools to target and detect extracellular ATP. We previously developed genetically-encoded fluorescent protein-based ATP sensors to monitor extracellular ATP levels. Currently, we are developing hybrid sensors in which one of the fluorescent proteins is replaced with a SNAP-tag protein. One hybrid sensor uses a turquoise fluorescent protein as a donor to a SNAP-tag acceptor. A different hybrid sensor uses a red fluorescent protein as an acceptor to a SNAP-tag donor. The linkers and N- versus C-terminal orientation of the SNAP-tag were optimized through small library screens. We are also modifying our hybrid sensors with different ATP binding domains to create variants with different ATP affinities. These hybrid sensors are characterized with spectroscopic, cell surface labeling, and ATP response experiments both with protein purified in solution and expressed in mammalian cells. This work was supported by NIH grant R01 GM134380 to M.T including a Diversity Supplement supporting A.S.