DEVELOPMENT OF "FRET-CAPTURE": A technique that exploits environmental-sensitive fluorophores as FRET donors to probe intermolecular protein-protein interactions and protein dynamics

“FRET-CAPTURE”的开发：利用环境敏感的荧光团作为 FRET 供体来探测分子间蛋白质-蛋白质相互作用和蛋白质动力学的技术

• 批准号: 194433412
• 负责人: Dr. Elke Socher
• 金额: --
• 依托单位: Department of Chemistry
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2011
• 资助国家: 德国
• 起止时间: 2010-12-31 至 2013-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/194433412?language=en
• 关键词: DEVELOPMENT; FRET; CAPTURE; technique; exploits

Fluorescence spectroscopy has become one of the most widely used tools for the development of new probes for ions, small molecules, and monitoring biological processes, such as protein folding, intracellular biomolecule distribution, protein-protein interactions, and phosphorylation events. An ideal approach for addressing these questions is the use of Förster resonance energy transfer (FRET), which is as one of the preferred methods for in cellulo studies. However, sometimes structural changes are too subtle to be detected with FRET. An alternative approach is the use of solvatochromic fluorophores which are a special class of molecules having spectroscopic properties that are dependent on the physicochemical properties of their environment. The group of Prof. Imperiali has developed new solvatochromic fluorophores as a powerful tool for the study of dynamic protein interactions. In this proposal the development of a fluorescence-based approach (FRET-CAPTURE) combining the principle of the solvatochromic fluorophores to induce energy transfer processes is described that could use the high apparent stokes shift of the FRET fluorescence for measurements with a high biological fluorescence background in living cells and could overcome the limitation of FRET.The initial model system for the methodology development will be based on the constructs that involve the calmodulin and the M13 peptide. Therefore several acceptor-donor combinations will be tested. Using the donor-acceptor combination showing the highest fluorescence increase for the FRET-CAPTURE signal it is planed to incorporate this tool in developing applications of the methodology for sensing various cytokines including EGF and TNF for application in 3D matrices.

荧光光谱已成为开发离子、小分子新探针和监测生物过程（例如蛋白质折叠、细胞内生物分子分布、蛋白质-蛋白质相互作用和磷酸化事件）最广泛使用的工具之一。解决这些问题的理想方法是使用福斯特共振能量转移 (FRET)，它是纤维素研究的首选方法之一。然而，有时结构变化过于细微，无法通过 FRET 检测到。另一种方法是使用溶剂化变色荧光团，这是一类特殊的分子，其光谱特性取决于其环境的物理化学特性。 Imperiali 教授小组开发了新的溶剂化显色荧光团，作为研究动态蛋白质相互作用的有力工具。在该提案中，描述了一种基于荧光的方法（FRET-CAPTURE）的开发，该方法结合了溶剂化显色荧光团的原理来诱导能量转移过程，该方法可以利用 FRET 荧光的高表观斯托克斯位移进行活细胞中高生物荧光背景的测量，并且可以克服 FRET 的局限性。该方法开发的初始模型系统将基于涉及钙调蛋白和 M13肽。因此，将测试几种受体-供体组合。使用显示 FRET-CAPTURE 信号最高荧光增加的供体-受体组合，计划将该工具纳入开发用于感测各种细胞因子（包括 EGF 和 TNF）的方法的应用中，以应用于 3D 矩阵。