Modified Photoproteins as Labels and Molecular Switches

描述（由申请人提供）：生物发光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。我们建议进一步扩大这些发光蛋白的应用，通过修改它们具有独特的生物发光特性。这项工作的目标之一是改变这些发光蛋白的发色团结合口袋内的电子和氢键网络，以改变其发射波长。这将通过将非天然氨基酸掺入水母发光蛋白结构中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。我们还建议模仿水母维多利亚中发生的自然现象，其中能量从水母发光蛋白转移到GFP导致发射绿色光。为此，我们建议通过将荧光团连接到靠近腔肠素结合口袋的水母发光蛋白上的独特位点来制备基于蛋白质的“人工水母”。这将允许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。可以在靶分析物存在下“打开”的“分子开关”将通过构建水母发光蛋白变体与特定分析物的结合蛋白之间的杂合蛋白来制备。通过将结合蛋白的基因插入水母发光蛋白的基因来构建杂合蛋白。在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个部分结合在一起，从而允许生物发光的发射。此外，“分子开关”将通过制备一种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发射。当靶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。上述修饰的发光蛋白在生物分析中的应用将通过开发在疾病诊断中重要的分析物面板的高灵敏度测定来证明。此外，基于具有不同发射波长的水母发光蛋白变体的这些测定将被并入微离心微流体平台中，这将导致在护理点诊断和HTPS中应用的多重分析。

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.