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: 8579142
• 负责人: BABAK BORHAN
• 金额: $ 39.28万
• 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/8579142
• 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学相关的过程中起着主要作用，包括天线蛋白在光合作用中吸收光，多种水生物种的荧光和磷光特性和视觉感知系统。蛋白质/发色团复合物还发现生物成像中的广泛且必不可少的效用是荧光蛋白融合标签。因此，对这些相互作用的基本和适用的理解是各种领域的广泛兴趣。用作灵感，自然系统会引起高级灵长类动物的色彩视觉，我们将 创建新型的蛋白质/发色团系统，并使用它们充分询问蛋白质和发色团之间的独特相互作用，从而产生该复合物的独特光谱特征。然后，我们将进一步开发这些系统，用于现实世界中的蛋白质可视化和体内pH传感。我们选择了彩色视觉系统来进行灵感，因为这里的自然表明，可以通过其蛋白质环境来调节单个配体色素团的吸收特性，以覆盖整个视觉光谱。在先前的研究中，我们已经证明，可以通过应用理性蛋白质设计策略在整个视觉范围内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时围绕发色团的静电环境来实现的。现在，我们将采用这些见解，并将其应用于蛋白质/荧光团复合物的合理设计，该蛋白质/荧光团复合物将调节荧光团的发射光谱。我们将专注于溶剂化荧光团，因为它们的发射光谱对溶剂极性非常敏感。 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/发色团复合物来严格研究。这些研究中开发的原理将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，这些蛋白质将在细菌和哺乳动物细胞类型的体外和体内作为融合蛋白进行测试。将开发荧光pH传感器以在各种细胞环境中的pH值进行体内测量。这些融合蛋白将在包括厌氧环境在内的各种应用中有用。它们也将用于对蛋白质检测的时间控制，其中仅检测在定义的时间窗口中表达的蛋白质。它们将共同代表当前可用的荧光蛋白的一组互补工具，并将大大扩展此可视化技术的有用性。