Modified Photoproteins as Labels and Molecular Switches

作为标签和分子开关的修饰光蛋白

• 批准号: 6880078
• 负责人: Sylvia Daunert
• 金额: $ 28.84万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-01-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6880078
• 关键词: bioluminescence; biomarker; calcium indicator; chimeric proteins; chromophore; fusion gene; green fluorescent proteins; hydrogen bond; immunologic assay /test; ligands; luciferin monooxygenase; method development; polymerase chain reaction; protein structure; protein structure function; reagent /indicator; site directed mutagenesis

DESCRIPTION (provided by applicant): Bioluminescent photoproteins afford high sensitivity of detection and have been employed as labels in bioanalysis. We propose to further expand the applications of these photoproteins by modifying them to possess unique bioluminescence properties. One of the goals of this work is to alter the electronic and H-bonding network within the chromophore-binding pocket of these photoproteins in order to shift their emission wavelengths. This will be achieved by incorporation of non-natural amino acids into the aequorin structure and by performing site-directed mutagenesis. The resulting proteins will be characterized in terms of their activity and structure-function relationship. We also propose to mimic the natural phenomenon that occurs in the jellyfish Aequorea Victoria where transfer of energy from aequorin to GFP results in the emission of green light. For that, we propose to prepare protein-based "artificial jellyfish" by attaching a fluorophore to unique sites on aequorin close to the coelenterazine binding pocket. This will allow transfer of energy from aequorin to the fluorophore, thus, shifting the wavelength of emission of the protein. "Molecular switches" that can be "turned on" in the presence of a target analyte will be prepared by constructing hybrid proteins between aequorin variants and a binding protein for a specific analyte. The hybrid proteins will be constructed by inserting the gene of the binding protein into the gene of aequorin. The conformational changes that occur in the binding protein upon ligand binding will bring the two parts of aequorin together, allowing for the emission of bioluminescence. In addition, "molecular switches" will be constructed by preparing fusion proteins of a dissected aequorin and genes that code for a pair of poplypeptides that can form a leucine zipper. The leucine zipper allows for aequorin to re-assembly and bioluminescence emission. When target DNA is present, the leucine zipper is pulled apart; aequorin is disassembled, with the subsequent loss of light. Applications of the above modified photoproteins in bioanalysis will be demonstrated by developing highly sensitive assays for panels of analytes that are important in disease diagnosis. Furthermore, these assays based on aequorin variants with different emission wavelengths will be incorporated into a microcentrifugal microfluidic platform, which should result in multiplex analysis with applications in point-of-care diagnostics and HTPS.

描述（由申请人提供）：生物发光蛋白提供高灵敏度的检测并已被用作生物分析中的标记。我们建议通过修饰这些发光蛋白以使其具有独特的生物发光特性，进一步扩大它们的应用。这项工作的目标之一是改变这些发光蛋白发色团结合口袋内的电子和氢键网络，以改变它们的发射波长。这将通过将非天然氨基酸掺入水母发光蛋白结构并通过进行定点诱变来实现。所得蛋白质将根据其活性和结构功能关系进行表征。我们还建议模仿维多利亚水母中发生的自然现象，其中能量从水母蛋白转移到 GFP 导致发射绿光。为此，我们建议通过将荧光团附着到靠近腔肠素结合袋的水母发光蛋白上的独特位点来制备基于蛋白质的“人造水母”。这将使能量从水母发光蛋白转移到荧光团，从而改变蛋白质的发射波长。通过构建水母发光蛋白变体和特定分析物的结合蛋白之间的杂合蛋白，可以制备在目标分析物存在的情况下“打开”的“分子开关”。通过将结合蛋白的基因插入到水母发光蛋白的基因中来构建杂合蛋白。配体结合时结合蛋白中发生的构象变化将使水母发光蛋白的两部分结合在一起，从而发射生物发光。此外，“分子开关”将通过制备切割的水母发光蛋白和编码一对可形成亮氨酸拉链的多肽的基因的融合蛋白来构建。亮氨酸拉链允许水母发光蛋白重新组装和生物发光。当目标DNA存在时，亮氨酸拉链被拉开；水母发光蛋白被分解，随后失去光。上述修饰发光蛋白在生物分析中的应用将通过开发对疾病诊断很重要的一组分析物的高灵敏度测定法来证明。此外，这些基于具有不同发射波长的水母发光蛋白变体的检测将被纳入微离心微流体平台中，这将导致多重分析并应用于现场诊断和HTPS。