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

荧光光谱已成为最广泛使用的工具之一，用于开发离子，小分子的新探针，并监测生物过程，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。解决这些问题的理想方法是使用Förster共振能量转移（FRET），这是在纤维素研究中首选的方法之一。然而，有时结构变化太微妙而无法用FRET检测到。另一种方法是使用溶剂致变色荧光团，这是一类特殊的分子，其光谱性质取决于其环境的物理化学性质。Imperiali教授小组开发了新的溶剂致变色荧光团，作为动态蛋白质相互作用研究的有力工具。在本提案中，基于荧光的方法（FRET- capture）的发展结合了溶剂致变色荧光团的原理来诱导能量转移过程，描述了可以利用FRET荧光的高表观斯托克斯位移在活细胞中具有高生物荧光背景的测量，并可以克服FRET的限制。方法论开发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检测各种细胞因子（包括EGF和TNF）的方法的应用中，用于3D矩阵。

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.