ATOMIC RESOLUTION STRUCTURES OF THE GREEN FLUORESCENT PROTEIN

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 我们建议：（i）继续研究绿色荧光蛋白（GFP）家族的翻译后化学和（ii）通过构建具有潜在应用的金属结合GFP变体作为体内生物传感器来测试基于算法的设计方法。GFP彻底改变了分子标记和细胞标记，包括在蛋白质运输、基因表达以及病原体功能和人类疾病研究中的应用。绿色荧光蛋白（及其同系物）非常适合高分辨率的结构，光谱，突变和计算研究，揭示在原子细节蛋白质如何自我合成发色团和调整发色团？化学和生物学功能的物理性质。通过高分辨率SSRL晶体学数据进行的初步研究使我们能够识别GFP中显著和前所未有的自发氨基酸修饰，包括氧掺入，肽键水解，氧化交联，脱羧和碳-碳键裂解反应。此外，可再现地获得变体晶体的能力使得GFP成为优异的设计靶支架。合理设计具有所需功能特性的金属蛋白具有巨大的生物技术或医学应用潜力。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，我们的目标是链接设计的金属网站的GFP发色团的荧光特性，以创建一个新的报告系统，允许在体内金属离子浓度的监测。使用多轮递归设计，通过在SSRL收集的高分辨率数据，我们已经创建了多个金属位点设计，调节GFP荧光特性。这些金属离子生物传感器的高分辨率结构分析，沿着它们的设计中间体和apo结构，使我们能够关闭设计周期并严格评估和

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: (i) continue investigating the post-translational chemistry for the green fluorescent protein (GFP) family and (ii) test algorithm-based design methodology through the construction of metal-binding GFP variants with potential applications as in vivo biosensors. GFP has revolutionized molecular tagging and cell labeling, including applications in protein trafficking, gene expression, and the study of pathogen function and human disease. 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. Initial studies made possible by high resolution SSRL crystallographic data allowed us to identify remarkable and unprecedented spontaneous amino acid modifications in GFP that include oxygen incorporation, peptide bond hydrolysis, oxidative cross links, decarboxylation, and carbon-carbon bond cleavage reactions. Moreover, the ability to reproducibly obtain crystals of variants makes GFP an excellent design target scaffold. The rational design of metalloproteins with desired functional properties has tremendous potential for biotechnological or medial 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 collected 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