Modified Photoproteins as Labels and Molecular Switches

描述(申请人提供)：生物发光光蛋白具有很高的检测灵敏度，已被用作生物分析的标记。我们建议通过对这些光蛋白进行修饰，使其具有独特的生物发光特性，从而进一步扩大其应用范围。这项工作的目标之一是改变这些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结合到aequorin结构中并通过执行定点突变来实现。由此产生的蛋白质将根据它们的活性和结构-功能关系进行表征。我们还建议模仿水母Aequorea Victoria中发生的自然现象，在这种现象中，能量从aequorin转移到GFP会导致绿色光的发射。为此，我们建议通过将荧光团附着在靠近coelenterazine结合口袋的aequorin上的独特位置来制备基于蛋白质的“人造水母”。这将允许能量从茶多酚转移到荧光团，从而改变蛋白质的发射波长。可以在目标分析物存在的情况下“开启”的“分子开关”将通过在aequorin变体和特定分析物的结合蛋白之间构建杂交蛋白来制备。通过将结合蛋白的基因插入到aequorin基因中，构建出杂交蛋白。当配体结合时，结合蛋白中发生的构象变化将使aequorin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”将通过制备分离的aequorin和编码一对能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aequorin重新组装和生物发光发射。当目标DNA存在时，亮氨酸拉链被拉开；aequorin被分解，随后失去光。上述修饰的光蛋白在生物分析中的应用将通过开发对疾病诊断重要的分析物面板的高灵敏度分析来展示。此外，这些基于不同发射波长的Aequorin变体的分析将被整合到一个微离心式微流控平台中，这将导致多重分析，并应用于医疗诊断和HTPS。

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.