Splitting GFP to look at signaling proteins

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DESCRIPTION (provided by applicant): This R21 proposal is to test a novel approach. The idea is compelling, and the approach could have a significant impact on many fields of basic research and biosensor development. Or it may not be feasible. The three proposed aims are designed to be direct tests of feasibility. The idea involves splitting a fluorescent protein and inserting the two fragments into different domains of a protein involved in cell signaling. The idea is that if the fluroescent protein is created betwee two subunits, or two different domains, it may be susceptible to relative movement of the proteins such that changes in fluorescence occurs. The goal is to create better reporters of cellular signaling in the nervous sytem. Such probes would offer us better temporal and spatial resolution. Moroever, they would enable us to optically record signaling activity in defined, genetically accessible, neural networks. The three aims of this proposal involve scanning a signaling molecule with a transposon tagging system that can insert fluorescent protein fragements into the target protein. In theory, a GFP should be formed at surfaces of the signaling protein that are close to one another. The hope is that these "split" GFPs may be more sensitive to the sort of distortion that will occur when protein domains move apart from one another. To test this idea, we will extensively scan a G protein subunit and two voltage-gated ion channels. The approach, experimental design, and scope of the project should suffice to show us whether this approach is worth further development.