ATOMIC RESOLUTION STRUCTURES OF THE GREEN FLUORESCENT PROTEIN

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We propose to investigate green fluorescent protein (GFP) chromophore self-synthesis and use algorithm-based design methodology to construct GFP metallomutants with applications as in vivo biosensors. GFP has revolutionized molecular tagging and cell labeling, including studies of protein trafficking, gene expression, and applications in human disease. Although the driving force and mechanism for GFP chromophore formation are not well understood, GFP (and its homologs) are well-suited for high-resolution structural, spectroscopic, mutational, and computational studies that reveal in atomic detail how proteins self-synthesize their chromophores and tune the chromophore¿s photophysical properties for chemical and biological function. Moreover, these experimental properties make GFP an excellent design target scaffold. The rational design of metalloproteins with desired functional properties has tremendous potential for biotechnological or medical applications. We are using an algorithm-based methodology (DEZYMER) to design metal-binding sites into GFP as a first step towards metalloprotein functional design. In addition, we aim to link the designed metal site to the fluorescent properties of the GFP chromophore to create a novel reporter system that permits monitoring of in vivo metal ion concentrations. Using rounds of recursive design, made possible by high-resolution data collection at SSRL, we have created multiple metal site designs that modulate GFP fluorescent properties. High resolution structural analysis of these metal ion biosensors, along with their design intermediates and apo structures, allow us to close the design cycle and rigorously evaluate and improve the DEZYMER algorithm. We believe these algorithm-designed biosensors and the ability to control and modify chromophore synthesis will have a major impact in the protein engineering and cell biology fields.