Protein/Chromphore Interactions via Protein Design: Interrogation and Application

描述(申请人提供)：蛋白质发色团相互作用在各种生物相关过程中发挥主要作用，包括光合作用中天线蛋白质对光的吸收，各种水生物种的荧光和磷光特性以及视觉感知系统。蛋白质/生色团复合体作为荧光蛋白质融合标签在生物成像中也发现了广泛而重要的用途。因此，对这些相互作用的基本和适用的理解对各种领域具有广泛的兴趣。利用高等灵长类动物产生颜色视觉的自然系统作为灵感，我们将 创建新的蛋白质/生色团系统，并使用它们来充分研究蛋白质和生色团之间的独特相互作用，从而产生复合体的独特光谱特征。然后，我们将进一步开发这些系统，用于蛋白质可视化和活体pH传感的现实世界应用。我们之所以选择色觉系统作为灵感来源，是因为在这里，大自然已经证明，单一配体发色团11-顺式视网膜的吸收特性可以受到其蛋白质环境的调节，覆盖整个视觉光谱。在以前的研究中，我们已经证明，通过应用合理的蛋白质设计策略，可以在整个视觉光谱中调节与蛋白质结合的视网膜的吸收光谱。这是通过控制嵌入到蛋白质环境中的发色团周围的静电环境来实现的。我们现在将利用这些见解，并将它们应用于合理设计蛋白质/荧光团复合体，以调节荧光团的发射光谱。我们将集中讨论溶剂变色荧光团，因为它们的发射光谱对溶剂的极性非常敏感。控制发色团pKA的因素也将通过产生具有广泛pKA值的蛋白质/发色团复合体来严格研究。这些研究中开发的原理将被应用于生产一类新的比色和荧光融合蛋白。这些蛋白质将表现出广泛的颜色、荧光激发和发射。将设计、合成和测试各种荧光配体，以进一步优化系统。然后，这些蛋白质将作为融合蛋白在体外和体内细菌和哺乳动物细胞类型中进行测试。荧光pH传感器将被开发用于在体内测量各种细胞环境中的pH。这些融合蛋白将在包括厌氧环境在内的各种应用中发挥作用。它们还将用于蛋白质检测的时间控制，其中将只检测在定义的时间窗口中表达的蛋白质。它们将共同代表一套补充目前可用的荧光蛋白质的工具，并将极大地扩展这种可视化技术的实用性。

DESCRIPTION (provided by applicant): Protein chromophore interactions play a major role in a variety of biologically relevant processes including the absorption of light by antenna proteins in photosynthesis, the fluorescent and phosphorescent properties of a variety of aquatic species and the visual perception system. Protein/chromophore complexes have also found wide and essential utility in biological imaging as fluorescent protein fusion tags. A fundamental and applicable understanding of these interactions is therefore of broad interest to a variety of field. Using as inspiration the natural system that gives rise to color vision in higher primates, we will create novel protein/chromophore systems and use them to fully interrogate the unique interplay between protein and chromophore that gives rise to the unique spectroscopic characteristics of the complex. We will then further develop these systems for real world applications in protein visualization and in vivo pH sensing. We have chosen the color vision system for inspiration because here nature has demonstrated that the absorption properties of a single ligand chromophore, 11-cis-retinal, can be modulated by its protein environment to cover the entire visual spectrum. In previous studies we have demonstrated that the absorption spectrum of protein-bound retinal can be modulated over the entire visual spectrum by the application of rational protein design strategies. This has been accomplished by controlling the electrostatic environment that surrounds the chromophore when embedded in the protein environment. We will now take these insights and apply them to the rational design of protein/fluorophore complexes that will modulate the emission spectrum of fluorophores. We will focus on solvatochromic fluorophores, because their emission spectra are exquisitely sensitive to the solvent polarity. The factors that govern the pKa of the chromophore will also be rigorously investigated by producing protein/chromophore complexes with a wide range of pKa values. The principles developed in these studies will be applied to produce a new class of colorimetric and fluorescent fusion proteins. These proteins will exhibit broad ranges of color, fluorescence excitation and emission. A variety of fluorescent ligands will be designed, synthesized and tested to further optimize the system. These proteins will then be tested as fusion proteins both in vitro and in vivo in bacterial and mammalian cell types. Fluorescent pH sensors will be developed for in vivo measurement of pH in a variety of cellular milieus. These fusion proteins will be useful in a variety of applications including anaerobic environments. They will also be useful for temporal control of protein detection, where only the protein expressed in a defined time window will be detected. Together they will represent a complementary set of tools to the fluorescent proteins currently available and will extend the usefulness of this visualization technique substantially.