Protein/Chromphore Interactions via Protein Design: Interrogation and Application

项目摘要 我们的计划是通过重新设计脂肪酸结合蛋白家族的成员来模拟 视紫红质，通过合理的设计原则。在实现这些目标的过程中，我们确信 这些极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。使用 它们体积小，可容纳各种无关结构的大结合口袋，高表达产率， 对突变导致的结构错误折叠的抵抗力，以及结晶倾向，我们发现这些蛋白质是理想的 传感器和成像应用工具。它们发展成为独特的荧光蛋白的故事还在继续 这项建议的结果是两个根本上重要的观察结果，转化为这项赠款的两个主要目标。 首先，在适当地设计了绑定口袋，并选择了适当的发色团作为合作伙伴后，我们 展示了蛋白质/发色团复合体作为亚胺的产生，它在光照射下经历 激发态质子转移(ESPT)来产生金属。设计发色团的关键是金属 质子化产生了能够进行分子内电荷转移(ICT)的高度共轭体系。ICT荧光团是 典型的红移和强荧光。因此，蓝色吸收络合物的光照射导致了激发的红色- 移动的物种，这种荧光具有很大的斯托克斯位移(LSS)。第二，我们已经意识到有能力创造一个 一套平行的可光切换的荧光染料，其中的荧光输出可以快速和光化学的 在‘开’和‘关’状态之间切换。这样的荧光系统是超高辐射所需的必要工具 分辨率显微镜，这项技术有可能彻底改变我们对生物现象的理解，如果 可以开发出合适的荧光染料。它们在生物成像应用中也是必不可少的，需要 时空控制。 实现这些目标的方法包括精确的、基于结构的设计和优化蛋白质和 找到了荧光团的理想体系。我们将优化蛋白质/生色团络合物作为光碱的ESPT，a 光酸，以及我们建议的双ESPT模式，需要光酸和光碱活性 在单次光激发事件中。这将把基态中性亚胺转化为两性离子，高度 极化的、共轭的激发态，将在远离激发波长的红光中发射。照片的设计- 切换与支持和促进结构变化的适当蛋白质环境结合的荧光团 在生色团中，会优化所需的特性，以获得所需的光开关，如红移 发射和高亮度。此外，“开”和“关”的动力学将被优化，因为快速的速率有利于 许多成像应用程序。

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.