An Instrument for Lanthanide-Luminescence Lifetime Microscopy

描述（由申请人提供）：构象变化和蛋白质相互作用是许多蛋白质功能的基础，但对于跨膜蛋白（如离子通道和G蛋白偶联受体）尤其重要，其跨膜传递信号。在结构水平上理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。 建议购买能够测量这种构象变化和精确测量蛋白质接近度的仪器。该仪器利用了发光镧系元素的独特性质，包括Tb 3+和Eu 3+。这些元素的发光的长ms寿命衰减可以高精度地测量，因此允许高精度地测量共振能量转移到附近的受体发色团（LRET）。寿命将从爪蟾卵母细胞中表达的蛋白质测量，镧系元素螯合物特异性地附着于它们。卵母细胞表达系统提供了可被检测的蛋白质的灵活性，以及在可接近的半胱氨酸取代的氨基酸上放置标记的灵活性。 该仪器可作为两个组件商购获得。第一组件包括用于激发镧系元素的光学器件、用于将激发聚焦在卵母细胞上的显微镜以及收集时间依赖性发光衰减的发射光学器件。将使用仪器提供的拟合软件分析衰变。第二个组件提供卵母细胞的电生理电压钳，并允许测量通过离子通道的电流。因此，我们获得了直接相关的功能变化与LRET测量的距离变化。 结构和功能变化的详细相关性将提供对膜蛋白功能的更高水平的理解。本申请中的许多感兴趣的蛋白质是治疗靶标。这项工作将提高我们将药物靶向特定蛋白质构象的能力，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.