ATOMIC RESOLUTION STRUCTURES OF THE GREEN FLUORESCENT PROTEIN

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我们建议研究绿色荧光蛋白(GFP)发色团的自我合成，并使用基于算法的设计方法来构建GFP金属突变体，并将其应用于体内生物传感器。GFP使分子标记和细胞标记发生了革命性变化，包括蛋白质运输、基因表达和在人类疾病中的应用研究。虽然绿色荧光蛋白(GFP)发色团的形成动力和机制还不是很清楚，但绿色荧光蛋白(及其同系物)非常适合于高分辨率的结构、光谱、突变和计算研究，这些研究揭示了蛋白质如何自我合成他们的发色团，并调整发色团的光物理性质来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靶向支架。合理设计具有所需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。我们正在使用一种基于算法的方法(DEZYMER)将金属结合位点设计成GFP，作为迈向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，创建一种新的报告系统，允许监测体内金属离子的浓度。利用SSRL的高分辨率数据收集实现的多轮递归设计，我们已经创建了多个调节GFP荧光特性的金属位点设计。对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，使我们能够缩短设计周期，并严格评估和改进DEZYMER算法。我们相信，这些由算法设计的生物传感器以及控制和修改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.