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: 8727065
• 负责人: BABAK BORHAN
• 金额: $ 29.28万
• 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: 2013-09-01 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8727065
• 关键词: 11 cis Retinal; Address; Affect; Affinity; Binding Sites; Biological; Biological Process; Breathing; Cell Line; Cells; Characteristics; Chimeric Proteins; Color; Color Visions; Complex; Detection; Development; Electrostatics; Environment; Eukaryotic Cell; Exhibits; Fluorescence; Goals; Green Fluorescent Proteins; Growth; Image; Imagery; In Vitro; Investigation; Knowledge; Lead; Left; Life; Ligand Binding; Ligands; Light; Location; Mammalian Cell; Measurement; Metal Binding Site; Monitor; Nature; Opsin; Output; Perception; Photosynthesis; Play; Primates; Process; Property; Protein Binding; Protein Engineering; Proteins; Regulation; Research; Resolution; Retinal; Role; Solvents; Structure; System; Techniques; Testing; Time; Variant; Visual; Visual Perception; Visual system structure; Whole Organism; Work; absorption; base; cell type; chromophore; design; fluorophore; in vivo; insight; interest; novel; polypeptide; programs; protein folding; protein structure; public health relevance; real world application; research study; sensor; success; tool

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.

描述（由申请人提供）：蛋白质发色团相互作用在各种生物相关过程中发挥重要作用，包括光合作用中天线蛋白对光的吸收、各种水生物种的荧光和磷光性质以及视觉感知系统。蛋白质/发色团复合物作为荧光蛋白融合标签在生物成像中也具有广泛和重要的用途。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色觉的自然系统作为灵感， 创建新的蛋白质/发色团系统，并使用它们来充分询问蛋白质和发色团之间的独特相互作用，从而产生复合物的独特光谱特性。然后，我们将进一步开发这些系统的真实的世界中的蛋白质可视化和体内pH传感的应用。我们之所以选择色觉系统作为灵感来源，是因为自然界已经证明，单一配体发色团11-顺式-视黄醛的吸收特性可以通过其蛋白质环境进行调节，以覆盖整个可见光谱。在以前的研究中，我们已经证明，蛋白质结合的视网膜的吸收光谱可以通过合理的蛋白质设计策略的应用在整个可见光谱上进行调制。这已经通过控制当嵌入蛋白质环境中时围绕发色团的静电环境来实现。我们现在将这些见解，并将其应用到蛋白质/荧光团复合物的合理设计，将调制荧光团的发射光谱。我们将集中在溶剂化显色荧光团，因为他们的发射光谱是非常敏感的溶剂极性。 还将通过产生具有宽范围pKa值的蛋白质/发色团复合物来严格研究支配发色团的pKa的因素。在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宽范围的颜色、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该系统。然后，这些蛋白质将作为融合蛋白在细菌和哺乳动物细胞类型中进行体外和体内测试。荧光pH传感器将被开发用于在各种细胞环境中的pH的体内测量。这些融合蛋白将可用于包括厌氧环境在内的多种应用。它们还将用于蛋白质检测的时间控制，其中仅在限定的时间窗口中表达的蛋白质将被检测。它们将共同代表一套互补的工具，目前可用的荧光蛋白，并将大大扩展这种可视化技术的有用性。