ATOMIC RESOLUTION STRUCTURES OF THE GREEN FLUORESCENT PROTEIN

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。我们建议研究绿色荧光蛋白（GFP）发色团的自合成，并使用基于算法的设计方法构建具有体内生物传感器应用的绿色荧光蛋白金属突变体。绿色荧光蛋白已经彻底改变了分子标记和细胞标记，包括蛋白质运输、基因表达和人类疾病应用的研究。虽然对GFP发色团形成的驱动力和机制尚不清楚，但GFP（及其同源物）非常适合用于高分辨率结构、光谱、突变和计算研究，这些研究可以从原子细节上揭示蛋白质如何自我合成其发色团，并调节发色团的光物理性质以实现化学和生物功能。此外，这些实验特性使GFP成为一种优秀的设计目标支架。合理设计具有理想功能特性的金属蛋白在生物技术或医学应用方面具有巨大的潜力。我们正在使用基于算法的方法（DEZYMER）来设计金属结合位点到GFP中，作为金属蛋白功能设计的第一步。此外，我们的目标是将设计的金属位点与GFP发色团的荧光特性联系起来，以创建一个新的报告系统，允许监测体内金属离子浓度。通过SSRL的高分辨率数据收集，我们使用了几轮递归设计，创建了多个调节GFP荧光特性的金属站点设计。这些金属离子生物传感器的高分辨率结构分析，以及它们的设计中间体和载脂蛋白结构，使我们能够关闭设计周期，严格评估和改进DEZYMER算法。我们相信这些算法设计的生物传感器以及控制和修饰发色团合成的能力将在蛋白质工程和细胞生物学领域产生重大影响。

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.