Protein/Chromphore Interactions via Protein Design: Interrogation and Application

项目摘要 我们通过重新设计脂肪酸结合蛋白家族的成员来开始我们的计划， 视紫红质，通过合理的设计原则。在实现这些目标的过程中，我们深信， 这些非常稳定的蛋白质的特性使它们成为各种其他应用的理想载体。与 它们体积小，结合口袋大，可容纳多种不相关的结构，表达产量高， 由于突变和结晶倾向，我们发现这些蛋白质是理想的 用于传感器和成像应用的工具。他们发展成独特的荧光蛋白的故事还在继续 这一建议是由于两个根本性的重要意见，转化为这一赠款的两个主要目标。 首先，适当地设计了结合口袋，并选择了适当的发色团作为伴侣， 证明了作为亚胺的蛋白质/发色团复合物的产生，其在光照射后经历 激发态质子转移（ESPT）以产生亚胺鎓。生色团设计的关键是亚胺鎓 质子化产生能够进行分子内电荷转移（ICT）的高度共轭体系。ICT荧光团是 通常是红移的和高荧光的。因此，蓝光吸收复合物的光照射导致激发的红光- 移动物种，其发出具有大斯托克斯位移（LSS）的荧光。第二，我们已经意识到创造一个 光可切换荧光染料的平行套件，其中荧光输出可以快速地光化学地 在“ON”和“OFF”状态之间切换。这样的荧光系统是用于超高分子量聚合物的必要工具。 分辨率显微镜，一种有可能彻底改变我们对生物现象的理解的技术， 可以开发出合适的荧光染料。它们在生物成像应用中也必不可少， 时空控制 实现这些目标的方法涉及蛋白质和蛋白质的精确的、基于结构的设计和优化。 荧光团，以找到理想的系统。我们将优化蛋白质/发色团复合物的ESPT作为光碱， 光酸，也在我们建议的双ESPT模式，需要光酸和光碱活性 在单个光激发事件期间。这将使基态中性亚胺转化为两性离子的高度亲核化亚胺。 偏振的共轭激发态，将发射远离激发波长的红光。照片的设计- 切换荧光团与支持和促进结构变化的适当蛋白质环境结合 将优化获得所需光开关所需的特性，例如红移的 发光和高亮度。此外，“开”和“关”动力学将被优化，因为快速的速率在以下方面是有利的： 许多成像应用。

Project Summary We began our program by re-engineering members of the Fatty Acid Binding protein family to be mimics of rhodopsin, through rational design principles. In the process of achieving these goals we became convinced that the characteristics of these fantastically stable proteins rendered them ideal vehicles for a variety of other applications. With their small size, large binding pocket that could accommodate a variety of unrelated structures, high expression yield, resistance to structural misfolding due to mutations, and propensity for crystallization, we found these proteins as ideal tools for sensor and imaging applications. The story of their development into unique fluorescent proteins continues with this proposal as a result of two fundamentally important observations, translated to the two major aims of this grant. First, having suitably engineered the binding pocket, and chosen the appropriate chromophore as a partner, we demonstrate the creation of a protein/chromophore complex as an imine, which upon photo-irradiation experiences Excited State Proton Transfer (ESPT) to generate the iminium. Critical to the design of the chromophore is that iminium protonation generates a highly conjugated system capable of Intramolecular Charge Transfer (ICT). ICT fluorophores are typically red-shifted and highly fluorescent. Thus, photo-irradiation of a blue absorbing complex leads to an excited red- shifting species, which fluoresces with a Large Stokes Shift (LSS). Second, we have realized the ability to create a parallel suite of photo-switchable fluorochromes, where the fluorescence output can be rapidly and photo-chemically switched between `ON' and `OFF' states. Such fluorescent systems are the essential tools required for ultra-high resolution microscopy, a technology that has the potential to revolutionize our understanding of biological phenomena if the proper fluorochromes can be developed. They are also essential in biological imaging applications that require spatio-temporal control. The approach to these goals involves the precise, structure-based design and optimization of both protein and fluorophore to find the ideal system. We will optimize ESPT of a protein/chromophore complex as a photobase, a photoacid, and also in what we suggest to be a dual-ESPT mode, requiring both photoacid and photobase activity during the single photo-excitation event. This would convert a ground state neutral imine to a zwitterionic, highly polarized, conjugated excited state that will emit far in the red from the wavelength of excitation. The design of photo- switching fluorophores married to the appropriate protein environment that supports and promotes the structural change in the chromophore, will optimize the characteristics necessary for obtaining a desired photo-switch, such as red-shifted emission and high brightness. Furthermore, `ON' and `OFF' kinetics will be optimized, as rapid rates are advantageous in many imaging applications.