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：(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于活体生物传感器的金属结合GFP变异体来测试基于算法的设计方法。GFP使分子标记和细胞标记发生了革命性的变化，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发色团的原子细节，并调整了生色团S的光物理性质，以实现化学和生物功能。通过高分辨率SSRL结晶学数据进行的初步研究使我们能够确定GFP中显著的和前所未有的自发氨基酸修饰，包括氧掺入、肽键水解、氧化交联键、脱羧基和碳-碳键断裂反应。此外，可重复获得变体晶体的能力使GFP成为优秀的设计目标支架。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收集的高分辨率数据，我们使用了几轮递归设计，创建了多个调节GFP荧光特性的金属位点设计。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，使我们能够结束设计周期，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