An Instrument for Lanthanide-Luminescence Lifetime Microscopy

描述(申请人提供)：构象变化和蛋白质相互作用是许多蛋白质功能的基础，但对跨膜蛋白质尤其关键，例如离子通道和G蛋白偶联受体，它们通过膜传递信号。在结构水平上了解这些变化对于合理开发针对这些蛋白质的未来疗法至关重要。建议购买一种能够测量这种构象变化和精确测量蛋白质近似性的仪器。该仪器利用了发光元素的独特性质，包括Tb3+和Eu3+。这些元素发光的长寿命(毫秒)衰减可以高精度地测量，因此可以高精度地测量到附近受主发色团(LRET)的共振能量转移。寿命将根据非洲爪哇卵母细胞中表达的蛋白质来测量，这些蛋白质上特别附着了稀土螯合物。卵母细胞表达系统在可检测的蛋白质和在可获得的半胱氨酸取代的氨基酸上放置标记方面提供了灵活性。该仪器以两个组件的形式在市场上出售。第一个组件包括用于激发稀土元素的光学元件、用于将激发聚焦到卵母细胞上的显微镜和用于收集随时间变化的发光衰减的发射光学元件。衰变将使用随仪器提供的拟合软件进行分析。第二部件为卵母细胞提供电生理电压钳，并允许测量通过离子通道的电流。因此，我们获得了功能变化与距离变化的LRET测量的直接关联。结构和功能变化的详细关联将提供对膜蛋白功能的更高水平的理解。在这一应用中，许多感兴趣的蛋白质都是治疗靶点。这项工作将提高我们针对特定蛋白质构象的药物靶向能力，从而提供高度特异性的药物作用。

DESCRIPTION (provided by applicant): Conformational changes and protein interactions underlie the function of many proteins, but are particularly critical for transmembrane proteins, such as ion channels and G-protein coupled receptors, which transfer signals across membranes. Understanding these changes at a structural level is essential for a rational development of future therapeutics that target these proteins. It is proposed to purchase an instrument capable of measuring such conformational changes and of precisely measuring proximity of proteins. The instrument takes advantage of the unique properties of the luminescent lanthanide elements, including Tb3+ and Eu3+. The long, ms, lifetime decays of the luminescence of these elements can be measured with high precision and therefore permits high-precision measurements of resonance energy transfer to nearby acceptor chromophores (LRET). The lifetimes will be measured from proteins expressed in Xenopus oocytes with lanthanide-chelates specifically attached to them. The oocyte expression system provides flexibility in the proteins that can be examined and in the placement of the labels on accessible, cysteine-substituted amino acids. The instrument is commercially available as two components. The first component comprises the optics for excitation of the lanthanides, a microscope for focusing the excitation on the oocyte, and emission optics, which collect time-dependent luminescence decays. The decays will be analyzed using fitting software provided with the instrument. The second component provides electrophysiological voltage clamp of the oocyte and permits measurement of currents through ion channels. Thereby, we obtain a direct correlation of functional changes with the LRET measurements of distance changes. A detailed correlation of structural and functional changes will provide a substantially higher level of understanding of membrane protein function. Many of the proteins of interest in this application are therapeutic targets. This work will improve our ability to target drugs to particular protein conformations and thereby deliver highly specific drug action.