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: 7012652
• 负责人: Sylvia Daunert
• 金额: $ 1.17万
• 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/7012652
• 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）：生物发光光蛋白具有高灵敏度的检测，并已在生物分析中用作标签。我们建议通过修饰它们具有独特的生物发光特性来进一步扩大这些光蛋白的应用。这项工作的目标之一是改变这些光蛋白的发色团结合袋中的电子和H键网络，以转移其发射波长。这将通过将非天然氨基酸掺入给火管结构并进行定位诱变来实现。所得的蛋白质将以其活性和结构功能关系来表征。我们还建议模仿在水母的eequorea victoria中发生的自然现象，其中能量从Aequorin转移到GFP会导致绿光的排放。为此，我们建议通过将荧光团连接到靠近腔室结合口袋的eequorin上的独特地点来制备基于蛋白质的“人造水母”。这将允许将能量从过溪转移到荧光团，从而改变蛋白质的发射波长。可以在存在目标分析物的情况下可以“打开”的“分子开关”，将通过在特定分析物之间构造equorin变体之间的杂化蛋白和结合蛋白。杂化蛋白将通过将结合蛋白的基因插入Aquorin的基因来构建。配体结合时结合蛋白中发生的构象变化将使eequorin的两个部分聚集在一起，从而允许发射生物发光。另外，将通过制备解剖的e equorin和基因的融合蛋白来构建“分子开关”，这些蛋白质和基因为一对可以形成亮氨酸拉链的普遍肽编码。亮氨酸拉链允许呼吸素重新组装和生物发光发射。当存在靶DNA时，将亮氨酸拉链拉开；释放eequorin被拆卸，随后的光损失。上述修饰的光蛋白在生物分析中的应用将通过为在疾病诊断中很重要的分析物的高度敏感测定中开发出高度敏感的测定。此外，这些基于具有不同发射波长的Aequorin变体的测定将被纳入微离心微流体平台中，这应该导致多重分析与服务点诊断和HTPS中的应用。