Protein/Chromphore Interactions via Protein Design: Interrogation and Application

通过蛋白质设计的蛋白质/发色团相互作用：询问和应用

• 批准号: 10197274
• 负责人: BABAK BORHAN
• 金额: $ 10.42万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-20 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10197274
• 关键词: Binding; Biological; Biological Phenomena; Characteristics; Charge; Chemicals; Complex; Crystallization; Development; Engineering; Environment; Event; Fluorescence; Fluorochrome; Goals; Grant; Image; Imines; Kinetics; Light; Microscopy; Mutation; Output; Process; Property; Protein Engineering; Protein Family; Proteins; Protons; Resistance; Rhodopsin; Structure; System; Technology; Translating; base; chromophore; design; experience; fatty acid-binding proteins; fluorophore; irradiation; live cell imaging; member; photo switch; programs; protonation; sensor; single molecule; spatiotemporal; tool; ultra high resolution

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.

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程序 Rhodopsin，通过合理的设计原则。在实现这些目标的过程中，我们确信 这些稳定的蛋白质的特征为它们提供了许多其他应用的理想车辆。和 它们的小尺寸，大的结合口袋，可以容纳各种无关的结构，高表达产量， 由于突变引起的结构错误折叠的抗性和结晶的倾向，我们发现这些蛋白质是理想的 传感器和成像应用的工具。他们开发成独特的荧光蛋白的故事仍在继续 这是由于两个根本重要的观察结果的结果，转化为这笔赠款的两个主要目标。 首先，在适当地设计了装订口袋，并选择了合适的发色团作为伴侣，我们 证明将蛋白质/发色团复合物作为亚胺的创建，在光辐射经验时 激发状态质子转移（ESPT）生成亚米属。发色团的设计至关重要 质子化产生了一个高度共轭系统，能够进行分子内电荷转移（ICT）。 ICT荧光团是 通常是红移且高度荧光的。因此，蓝色吸收复合物的光辐照导致激发的红色 转移物种，其荧光大大旋转（LSS）。其次，我们已经意识到能够创建一个 平行的光切换荧光色素套件，荧光输出可以迅速和光化。 切换在“ ON”和“ OFF”状态之间。这样的荧光系统是超高的必需工具 解决显微镜，该技术有可能彻底改变我们对生物现象的理解 可以开发适当的荧光色素。它们在需要的生物成像应用中也是必不可少的 时空对照。 实现这些目标的方法涉及蛋白质和蛋白质的精确，基于结构的设计和优化 荧光团找到理想的系统。我们将优化蛋白质/发色团复合物的ESPT作为光酶A 光acid，以及我们建议是双eSPT模式，需要光酸和光碱活性 在单次示威活动中。这会将基态中性亚胺转化为zwitterionic，高度 偏振，共轭激发态将与激发波长相去甚远。照片的设计 - 转换荧光团已婚，该环境已与适当的蛋白质环境结合在一起，该环境支持和促进结构变化 在发色团中，将优化获得所需的照片开关所需的特征，例如红移 排放和高亮度。此外，将优化“ ON”和“ OFF”动力学，因为快速速率在 许多成像应用。