Expanding the fluorescent toolkit with non-canonical amino acids

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大，但目前的方法在报告荧光的能力方面往往受到限制。 动态事件（如蛋白质-蛋白质相互作用、配体结合或细胞中金属离子的流动）， 大多数生物过程。拟议的研究旨在通过结合 计算蛋白质设计方法与技术允许细胞掺入荧光 蛋白质中的非典型氨基酸（fNCAAs），以产生新型的基于蛋白质的荧光 具有增强性能的传感器。 我们的努力将集中在开发新的基于蛋白质的工具，其中环境敏感的荧光团- 那些其荧光特性响应于周围环境的变化而改变的化合物， 作为动态细胞过程的传感器。使用这种染料研究细胞内动态过程的能力 环境中的荧光通常受到荧光团通常附着在靶表面的事实的限制 生物分子或者，将遗传编码的NCAA直接掺入肽骨架中， 因此，它们特别适合于对蛋白质支架内的细微变化做出反应。这表明fNCAAs 可以作为一个平台，用于创建一类新的基于蛋白质的传感器， 一系列的刺激。然而，实现这一目标需要能够确定fNCAA的最佳位点， 掺入使得预期荧光的明确变化而不破坏天然蛋白质 功能最近，我们在结构上表征了含有fNCAA和7-羟基香豆素（7-HC）的蛋白质。 对蛋白质-蛋白质和蛋白质-小分子相互作用有反应侧链。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 从而为合理设计蛋白质-小分子荧光生物传感器奠定了基础 交互.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，将用于工程小分子代谢物的新荧光传感器。在 第二个目标，我们将开发基于含有7-HC或8-HC的NCAAs的高选择性金属离子传感器。 羟基喹啉（8-HQ）作为侧链。同样，计算蛋白质设计方法将被用来塑造 这些NCAAs周围的蛋白质环境，以产生新的荧光蛋白， 生物学相关的金属浓度范围很广，并且可以选择性地在细胞中产生， 时空控制的方式最后，由于许多现有的fNCAA表现出荧光性质， 我们将扩大现有荧光NCAAs的工具包， 那些具有增强性质的分子，将有助于直接研究细胞中的生物过程。

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.