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着重要作用，包括光合作用中天线蛋白的光吸收、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/发色团复合物也作为荧光蛋白融合标签在生物成像中具有广泛且重要的用途。因此，对这些相互作用的基本和适用的理解引起了各个领域的广泛兴趣。以高等灵长类动物产生色觉的自然系统为灵感，我们将 创建新颖的蛋白质/发色团系统，并使用它们来充分探究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征。然后，我们将进一步开发这些系统，用于蛋白质可视化和体内 pH 传感的实际应用。我们选择色觉系统作为灵感，因为大自然已经证明，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，以覆盖整个视觉光谱。在之前的研究中，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上调节蛋白质结合的视网膜的吸收光谱。这是通过控制嵌入蛋白质环境中发色团周围的静电环境来实现的。现在，我们将利用这些见解并将其应用于蛋白质/荧光团复合物的合理设计，以调节荧光团的发射光谱。我们将重点关注溶剂致变色荧光团，因为它们的发射光谱对溶剂极性非常敏感。 还将通过生产具有广泛 pKa 值的蛋白质/发色团复合物来严格研究控制发色团 pKa 的因素。这些研究中开发的原理将用于生产一类新型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。多种荧光配体将被设计、合成和测试，以进一步优化系统。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测量各种细胞环境中的 pH 值。这些融合蛋白可用于多种应用，包括厌氧环境。它们还可用于蛋白质检测的时间控制，其中仅检测在定义的时间窗口内表达的蛋白质。它们将共同代表目前可用的荧光蛋白的一组补充工具，并将大大扩展这种可视化技术的实用性。