Knowledge-based design of a biosensor to quantify localized ERK activation in living cells.
Knowledge-based design of a biosensor to quantify localized ERK activation in living cells.
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DOI：
10.1016/j.chembiol.2013.04.016
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发表时间：
2013-06-20
影响因子：
--
通讯作者：
Plückthun A
中科院分区：
文献类型：
--
作者：
Kummer L;Hsu CW;Dagliyan O;MacNevin C;Kaufholz M;Zimmermann B;Dokholyan NV;Hahn KM;Plückthun A
研究活细胞中的蛋白质活化对于了解蛋白质如何受到它们所经历的上游调节剂的全部影响至关重要。我们在这里描述了一种生物传感器的基础上设计锚蛋白重复蛋白（DARPin）的结合支架适合于细胞内的应用。结合从文库中的选择和进化，基于知识的设计和缀合物候选物的有效和快速测试，我们通过用溶剂化显色mercury染料（mero 87）衍生化特异于磷酸化ERK（pERK）的DARPin来创建ERK活性生物传感器，其荧光在pERK结合后增加。该生物传感器特异性地响应pERK 2，通过其构象识别，但不响应非磷酸化ERK 2或其他密切相关的丝裂原活化激酶。在2%血清中孵育的小鼠胚胎成纤维细胞中可见激活的内源性ERK，揭示了细胞核、核周区域，尤其是核仁中的更大激活。活性被MEK 1/2抑制剂U 0126大大降低。基于DARPin的生物传感器将为研究ERK在体外和体内的生物学功能提供有用的工具。
Investigation of protein activation in living cells is fundamental to understand how proteins are influenced by the full complement of upstream regulators they experience. We describe here the generation of a biosensor based on the Designed Ankyrin Repeat Protein (DARPin) binding scaffold suited for intracellular applications. Combining selection and evolution from libraries, knowledge-based design and efficient and rapid testing of conjugate candidates, we created an ERK activity biosensor by derivatizing a DARPin specific for phosphorylated ERK (pERK) with a solvatochromic merocyanine dye (mero87), whose fluorescence increases upon pERK binding. The biosensor specifically responded to pERK2, recognized by its conformation, but not to non-phosphorylated ERK2 or other closely related mitogen-activated kinases tested. Activated endogenous ERK was visualized in mouse embryo fibroblasts incubated in 2% serum, revealing greater activation in the nucleus, perinuclear regions, and especially the nucleoli. Activity was greatly reduced by the MEK1/2 inhibitor U0126. The DARPin-based biosensor will serve as useful tool for studying biological functions of ERK in vitro and in vivo.
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影响因子：
5.6
作者：
Binz, HK;Stumpp, MT;Plückthun, A
通讯作者：
Plückthun, A
DOI：
10.1016/s1359-0278(98)00072-8
发表时间：
1998-01-01
期刊：
FOLDING & DESIGN
影响因子：
--
作者：
Dokholyan, NV;Buldyrev, SV;Shakhnovich, EI
通讯作者：
Shakhnovich, EI
影响因子：
4.8
作者：
Kawe, Martin;Forrer, Patrik;Plueckthun, Andreas
通讯作者：
Plueckthun, Andreas
影响因子：
4.8
作者：
Favata, MF;Horiuchi, KY;Trzaskos, JM
通讯作者：
Trzaskos, JM
影响因子：
5.7
作者：
Dagliyan, Onur;Proctor, Elizabeth A.;D'Auria, Kevin M.;Ding, Feng;Dokholyan, Nikolay V.
通讯作者：
Dokholyan, Nikolay V.