Expanding the fluorescent toolkit with non-canonical amino acids

使用非规范氨基酸扩展荧光工具包

• 批准号: 10377964
• 负责人: Jeremy Mills
• 金额: $ 34.09万
• 依托单位: ARIZONA STATE UNIVERSITY-TEMPE CAMPUS
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10377964
• 关键词: Achievement; Address; Affect; Affinity; Algorithm Design; Amino Acids; Binding Proteins; Biological Assay; Biological Process; Biology; Biosensor; Cell physiology; Cells; Cellular biology; Computing Methodologies; Coumarins; Data; Development; Dissociation; Dyes; Engineering; Environment; Equipment; Event; Exhibits; Fluorescence; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Foundations; Goals; Image; Industrialization; Ions; Ligand Binding; Ligands; Metalloproteins; Metals; Methods; Modeling; Modernization; Molecular Conformation; Nature; Oxyquinoline; Peptides; Positioning Attribute; Process; Property; Protein Engineering; Proteins; Reporter; Reporting; Research; Scaffolding Protein; Side; Site; Specificity; Stimulus; Surface; System; Techniques; Technology; Translating; Tryptophan; Umbelliferones; Vertebral column; base; biological systems; design; falls; flexibility; fluorophore; functional group; in vivo; in vivo imaging; insight; metal chelator; novel; protein function; protein protein interaction; rational design; response; sensor; small molecule; spatiotemporal; success; tool

PROJECT SUMMARY Fluorescent methods have revolutionized our ability to study biological processes in cellular environments. Despite an ever-expanding fluorescent toolkit, current methods are often limited in their ability to report on dynamic events (e.g. protein-protein interactions, ligand binding, or the flux of metal ions in cells) that underpin a majority of biological processes. The proposed research seeks to address this challenge by combining computational protein design methods with technology allowing the cellular incorporation of fluorescent non-canonical amino acids (fNCAAs) in proteins to generate novel classes of protein-based fluorescent sensors with enhanced properties. Our efforts will focus on developing new protein-based tools in which environmentally sensitive fluorophores— those whose fluorescent properties are modified in response to changes in the surrounding environment—serve as sensors of dynamic cellular processes. The ability to use such dyes to study dynamic processes in cellular environments is often limited by the fact that fluorophores are generally attached to the surfaces of target biomolecules. Alternatively, genetically encoded NCAAs are incorporated directly in the peptide backbone and are therefore uniquely suited to respond to subtle changes within protein scaffolds. This suggests that fNCAAs could serve as a platform for the creation of a novel class of protein-based sensors that dynamically respond to a host of stimuli. However, achievement of this goal would require the ability to identify optimal sites of fNCAA incorporation such that a well-defined change in fluorescence is expected without disrupting natural protein function. We recently structurally characterized proteins containing an fNCAA with a 7-hydroxycoumarin (7-HC) side chain that are responsive to protein-protein and protein-small molecule interactions. These data provide insight into how changes in the environment surrounding the 7-HC fluorophore translate into changes in its spectrum, thereby paving the way for the rational design of fluorescent biosensors of protein-small molecule interactions. To explore this possibility, our recently obtained structural data will serve as inputs to computational protein design methods that will be used to engineer new fluorescent sensors of small molecule metabolites. In a second aim, we will develop highly selective metal ion sensors based on NCAAs containing either 7-HC or 8- hydroxyquinoline (8-HQ) as a side chain. Again, computational protein design methods will be used to sculpt the protein environments surrounding these NCAAs in order to generate new fluorescent proteins that are sensitive to a wide range of biologically relevant metal concentrations and can be selectively produced in cells in a spatiotemporally controlled fashion. Finally, because many existing fNCAAs exhibit fluorescent properties that are not readily amenable to cell-based assays we will expand the toolkit of existing fluorescent NCAAs to include those with enhanced properties that will facilitate the direct study of biological processes in cells.

项目摘要 荧光方法彻底改变了我们研究细胞环境中生物过程的能力。 尽管荧光工具包不断扩展，但当前方法的报告能力通常受到限制 动态事件（例如蛋白质 - 蛋白质相互作用，配体结合或细胞中金属离子的通量） 大多数生物过程。拟议的研究旨在通过结合来应对这一挑战 使用技术的计算蛋白设计方法允许荧光掺入细胞 蛋白质中的非典型氨基酸（FNCAA），以生成新的基于蛋白质的荧光类 具有增强性能的传感器。 我们的努力将着重于开发基于蛋白质的新工具，在这种工具中，对环境敏感的荧光团（ 那些因周围环境变化而修改荧光性能的人 - 作为动态细胞过程的传感器。使用此类染料研究细胞中动态过程的能力 环境通常受到荧光团通常连接到目标表面的事实的限制 生物分子。或者，遗传编码的NCAA直接掺入胡椒骨架，并 因此，非常适合应对蛋白质支架内的细微变化。这表明fncaas 可以作为创建新型基于蛋白质的传感器的平台，这些传感器动态响应 一系列刺激。但是，实现这一目标将需要能够识别FNCAA的最佳站点 掺入使得荧光明确的变化无需破坏天然蛋白质 功能。我们最近在结构上表征了含有7-羟基丙酸酯（7-HC）的FNCAA的蛋白质 对蛋白质 - 蛋白质和蛋白质 - 小分子相互作用的副链链。这些数据提供 深入了解7-HC荧光团周围环境的变化如何转化为其变化 频谱，从而掩盖了蛋白质 - 小分子荧光生物传感器的合理设计的道路 互动。为了探索这种可能性，我们最近获得的结构数据将作为计算的输入 蛋白质设计方法将用于设计小分子代谢产物的新荧光传感器。 第二个目标，我们将基于含有7-HC或8--的NCAA开发高选择性的金属离子传感器 羟喹啉（8-hq）作为侧链。同样，计算蛋白设计方法将用于雕刻 这些NCAA周围的蛋白质环境，以生成敏感的新荧光蛋白 到广泛的生物学相关金属浓度，可以选择在A中产生细胞 时空控制的时尚。最后，因为许多现有的FNCAA暴露了荧光特性 不容易适合基于细胞的测定法，我们将扩展现有荧光NCAA的工具包 那些具有增强性能的人，可以促进细胞中生物过程的直接研究。