A Sensitized Emission Based Calibration of FRET Efficiency for Probing the Architecture of Macromolecular Machines.
A Sensitized Emission Based Calibration of FRET Efficiency for Probing the Architecture of Macromolecular Machines.
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DOI：
10.1007/s12195-013-0290-y
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发表时间：
2013
影响因子：
2.8
通讯作者：
Lawrimore, Joshua
中科院分区：
文献类型：
--
作者：
Joglekar, Ajit;Chen, Renjie;Lawrimore, Joshua
大分子机器参与几乎所有细胞的生物学功能。这些机器可以采取明确的蛋白质结构的形式，如动粒，或更松散的组织蛋白质组装，如内吞外套。这些机器的蛋白质结构-在纳米尺度上的多个蛋白质亚基拷贝的排列，对于理解它们的细胞生物学功能和生物物理机制是必要的。在体内定义这种架构是一个重大挑战。大分子机器内的高密度蛋白质分子严重限制了超分辨率显微镜的有效性。然而，这种密度对于福斯特共振能量转移（FRET）是理想的，它可以确定相邻分子之间的接近度。在这里，我们提出了一个简单的FRET定量方案，校准标准的落射荧光显微镜测量供体-受体分离。该校准可用于推导FRET效率荧光强度测量。该方法将允许在宽范围的值和FRET对数目上准确地确定FRET效率。它还将允许在细胞生物学条件下具有高时空分辨率的动态FRET测量。虽然遗传编码的荧光蛋白的成熟效率差提出了一个挑战，我们表明，它的影响可以减轻。为了证明这种方法，我们探测γ-微管蛋白环的体内结构。我们的技术可以应用于研究各种大分子机器的结构和动力学。本文的在线版本（doi：10.1007/s12195-013-0290-y）包含补充材料，可供授权用户使用。
Macromolecular machines participate in almost every cell biological function. These machines can take the form of well-defined protein structures such as the kinetochore, or more loosely organized protein assemblies like the endocytic coat. The protein architecture of these machines—the arrangement of multiple copies of protein subunits at the nanoscale, is necessary for understanding their cell biological function and biophysical mechanism. Defining this architecture in vivo presents a major challenge. High density of protein molecules within macromolecular machines severely limits the effectiveness of super-resolution microscopy. However, this density is ideal for Forster Resonance Energy Transfer (FRET), which can determine the proximity between neighboring molecules. Here, we present a simple FRET quantitation scheme that calibrates a standard epifluorescence microscope for measuring donor–acceptor separations. This calibration can be used to deduce FRET efficiency fluorescence intensity measurements. This method will allow accurate determination of FRET efficiency over a wide range of values and FRET pair number. It will also allow dynamic FRET measurements with high spatiotemporal resolution under cell biological conditions. Although the poor maturation efficiency of genetically encoded fluorescent proteins presents a challenge, we show that its effects can be alleviated. To demonstrate this methodology, we probe the in vivo architecture of the γ-Tubulin Ring. Our technique can be applied to study the architecture and dynamics of a wide range of macromolecular machines. The online version of this article (doi:10.1007/s12195-013-0290-y) contains supplementary material, which is available to authorized users.
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影响因子：
64.5
作者：
Kaksonen, M;Toret, CP;Drubin, DG
通讯作者：
Drubin, DG
DOI：
10.1016/s0075-7535(08)00001-6
发表时间：
2009-01-01
期刊：
FRET AND FLIM TECHNIQUES
影响因子：
--
作者：
Clegg, Robert M.
通讯作者：
Clegg, Robert M.
影响因子：
3.3
作者：
Albertazzi, Lorenzo;Arosio, Daniele;Beltram, Fabio
通讯作者：
Beltram, Fabio
影响因子：
64.5
作者：
Shivaraju, Manjunatha;Unruh, Jay R.;Gerton, Jennifer L.
通讯作者：
Gerton, Jennifer L.
影响因子：
3.4
作者：
Padilla-Parra, Sergi;Auduge, Nicolas;Tramier, Marc
通讯作者：
Tramier, Marc