Calcium Signals Laboratory, Department of Biomedical Engineering, The Johns Hopkins University School of Medicine, Ross Building, Room 713, 720 Rutland Avenue, Baltimore, Maryland 21205, USA.
Nat Commun. 2016 Dec 6;7:13709. doi: 10.1038/ncomms13709.
The stoichiometry of macromolecular interactions is fundamental to cellular signalling yet challenging to detect from living cells. Fluorescence resonance energy transfer (FRET) is a powerful phenomenon for characterizing close-range interactions whereby a donor fluorophore transfers energy to a closely juxtaposed acceptor. Recognizing that FRET measured from the acceptor's perspective reports a related but distinct quantity versus the donor, we utilize the ratiometric comparison of the two to obtain the stoichiometry of a complex. Applying this principle to the long-standing controversy of calmodulin binding to ion channels, we find a surprising Ca-induced switch in calmodulin stoichiometry with Ca channels-one calmodulin binds at basal cytosolic Ca levels while two calmodulins interact following Ca elevation. This feature is curiously absent for the related Na channels, also potently regulated by calmodulin. Overall, our assay adds to a burgeoning toolkit to pursue quantitative biochemistry of dynamic signalling complexes in living cells.
大分子相互作用的化学计量学对于细胞信号转导至关重要，但从活细胞中检测却具有挑战性。荧光共振能量转移（FRET）是一种强大的技术，可用于表征近距离相互作用，其中供体荧光团将能量转移到紧密相邻的受体上。我们认识到，从受体角度测量的 FRET 报告的是与供体相关但不同的数量，因此我们利用两者的比率比较来获得复合物的化学计量。将这一原理应用于钙调蛋白与离子通道结合的长期争议中，我们发现钙调蛋白与钙通道的结合存在一个令人惊讶的 Ca 诱导开关——在基础细胞浆 Ca 水平下结合一个钙调蛋白，而在 Ca 升高后则相互作用两个钙调蛋白。这种特征对于相关的钠通道来说是奇怪的缺失，钠通道也受到钙调蛋白的强烈调节。总的来说，我们的测定方法为在活细胞中研究动态信号复合物的定量生物化学增加了一个蓬勃发展的工具包。
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