Monitoring a coordinated exchange process in a four-component biological interaction system: development of a time-resolved terbium-based one-donor/three-acceptor multicolor FRET system.
Monitoring a coordinated exchange process in a four-component biological interaction system: development of a time-resolved terbium-based one-donor/three-acceptor multicolor FRET system.
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DOI：
10.1021/ja100248q
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发表时间：
2010-04-07
影响因子：
15
通讯作者：
Katzenellenbogen, John A.
中科院分区：
文献类型：
--
作者：
Kim, Sung Hoon;Gunther, Jillian R.;Katzenellenbogen, John A.
细胞功能的激素调节涉及小分子与受体的结合，这些受体随后协调与其他信号转导蛋白的后续相互作用。这些动态的、多组分的过程在细胞中甚至在重组的体外系统中都难以追踪，并且大多数方法只能监测双组分相互作用，通常在追踪动态变化方面能力有限。通过明智地选择三种有机受体荧光团与一种铽供体荧光团配对，我们开发了首个单供体/三受体多色时间分辨荧光能量转移（TR - FRET）系统的实例，并通过监测一个四组分生物系统中配体调节的蛋白质 - 蛋白质交换过程展示了其用途。通过仔细量化铽供体发射的四个通道中每个受体的发射，我们证明在这个铽供体 - 三受体系统中，这些供体通道中的任何一个都可用于估计三个FRET信号的强度，且串扰极小。使用这种基于铽的三通道TR - FRET检测系统，我们在一次实验中表明，添加一种荧光素标记的雌激素激动剂会使一种SNAPFL标记的抗雌激素从铽标记的雌激素受体的配体结合口袋中置换出来，同时导致一种Cy5标记的辅激活因子被招募到雌激素受体上。这个实验展示了四色TR - FRET实验的强大功能，并表明雌激素受体配体交换和辅激活因子结合的整个过程是一个动态但精确协调的过程。
Hormonal regulation of cellular function involves the binding of small molecules with receptors that then coordinate subsequent interactions with other signal transduction proteins. These dynamic, multi-component processes are difficult to track in cells and even in reconstituted in vitro systems, and most methods can monitor only two-component interactions, often with limited capacity to follow dynamic changes. Through a judicious choice of three organic acceptor fluorophores paired with a terbium donor fluorophore, we have developed the first example of a one-donor/three-acceptor multi-color time-resolved fluorescence energy transfer (TR-FRET) system, and we have exemplified its use by monitoring a ligand-regulated protein-protein exchange process in a four-component biological system. By careful quantification of the emission from each of the three acceptors at the four channels for terbium donor emission, we demonstrate that any of these donor channels can be used to estimate the magnitude of the three FRET signals in this terbium donor triple-acceptor system with minimal bleedthrough. Using this three-channel terbium-based, TR-FRET assay system, we show in one experiment that the addition of a fluorescein-labeled estrogen agonist displaces a SNAPFL-labeled antiestrogen from the ligand binding pocket of a terbium-labeled estrogen receptor, at the same time causing a Cy5-labeled coactivator to be recruited to the estrogen receptor. This experiment demonstrates the power of a four-color TR-FRET experiment, and it shows that the overall process of estrogen receptor ligand exchange and coactivator binding is a dynamic but precisely coordinated process.
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影响因子：
3.2
作者：
Poole, RA;Bobba, G;Peacock, RD
通讯作者：
Peacock, RD
影响因子：
1.8
作者：
Robers, Matthew B.;Machleidt, Thomas;Bi, Kun
通讯作者：
Bi, Kun
DOI：
10.1073/pnas.0607385103
发表时间：
2006-10-24
影响因子：
11.1
作者：
Paulmurugan, Ramasanny;Gambhir, Sanjiv S.
通讯作者：
Gambhir, Sanjiv S.
影响因子：
2.9
作者：
Jeyakumar, M.;Katzenellenbogen, John A.
通讯作者：
Katzenellenbogen, John A.
影响因子：
4.1
作者：
Quaedackers, Monique E.;van den Brink, Christina E.;Tertoolen, Leon G. J.
通讯作者：
Tertoolen, Leon G. J.