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

荧光光谱已经成为开发新的离子、小分子探针和监测生物过程(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)的最广泛使用的工具之一。解决这些问题的理想方法是使用Förster共振能量转移(FRET)，这是纤维素酶研究的首选方法之一。然而，有时结构变化太微妙，无法用FRET检测到。另一种方法是使用溶剂变色荧光团，这是一类特殊的分子，具有依赖于其环境的物理化学性质的光谱性质。Imperiali教授的团队开发了新的溶剂变色荧光团，作为研究动态蛋白质相互作用的有力工具。在这项建议中，描述了一种基于荧光的方法(FRET-Capture)的发展，该方法结合了溶剂变色荧光团诱导能量转移的原理，可以利用FRET荧光的高表观斯托克斯位移来测量活细胞中具有高生物荧光背景的测量，并可以克服FRET的局限性。方法学发展的初始模型系统将基于涉及钙调蛋白和M13肽的结构。因此，将对几种受体-供体组合进行测试。利用对FRET捕获信号显示最高荧光增加的供体-受体组合，计划将该工具应用于开发用于检测包括EGF和TNF在内的各种细胞因子的方法学在3D基质中的应用。

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.