Expanding the fluorescent toolkit with non-canonical amino acids

项目总结 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，但当前的方法往往在报告 作为基础的动态事件(例如，蛋白质-蛋白质相互作用、配体结合或细胞内金属离子的通量) 大多数生物过程。拟议的研究试图通过结合以下几个方面来应对这一挑战 允许细胞内掺入荧光的计算蛋白质设计方法 蛋白质中的非正则氨基酸(FNCAA)产生新型蛋白质荧光 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- 那些荧光特性随着周围环境的变化而改变的服务 作为动态细胞过程的传感器。使用这种染料研究细胞中的动态过程的能力 环境通常受到这样一个事实的限制，即荧光团通常附着在靶材表面 生物分子。或者，基因编码的NCAA直接结合在多肽骨架中，并 因此，它们非常适合于对蛋白质支架内的细微变化做出反应。这表明FNCAAs 可以作为一个平台来创造一种新型的基于蛋白质的传感器，它可以动态地响应 一大堆刺激。然而，实现这一目标需要有能力确定fNCAA的最佳地点 掺入，从而在不破坏天然蛋白质的情况下预期荧光的明确变化 功能。我们最近对含有fNCAA和7-羟基香豆素(7-HC)的蛋白质进行了结构表征 响应蛋白质-蛋白质和蛋白质-小分子相互作用的侧链。这些数据提供了 洞察7-HC荧光团周围环境的变化如何转化为其 从而为蛋白质小分子荧光生物传感器的合理设计奠定了基础 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 将用于设计新的小分子代谢物荧光传感器的蛋白质设计方法。在……里面 第二个目标，我们将开发基于含有7-HC或8-HC的NCAA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雕刻 这些NCAA周围的蛋白质环境，以便产生新的敏感的荧光蛋白 到广泛的生物相关金属浓度，可以在细胞中选择性地产生 时空控制的时尚。最后，由于许多现有的fNCAA显示出荧光特性， 不适合基于细胞的分析，我们将扩展现有荧光NCAA的工具包，以包括 它们具有增强的特性，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.