Deciphering the molecular mechanism responsible for GCaMP6m's Ca2+-dependent change in fluorescence.
Deciphering the molecular mechanism responsible for GCaMP6m's Ca2+-dependent change in fluorescence.
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DOI：
10.1371/journal.pone.0170934
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Drobizhev M
中科院分区：
文献类型：
--
作者：
Barnett LM;Hughes TE;Drobizhev M
这项工作的目标是确定GCaMP6m的荧光是如何响应Ca2+结合而改变的。我们详细的光谱研究揭示了GCaMP6m如何响应于Ca2+改变荧光的最简单的解释是四态模型，其中由于pKa的变化，发色团质子化状态的Ca2+依赖性变化是主要因素。的pKa位移定量解释的发色团周围的静电势的变化，由于发生在蛋白质中的构象变化时，钙调蛋白结合Ca2+和M13肽相互作用。GCaMP6m的无Ca2+和Ca2+饱和状态的绝对pKa值对其高信噪比至关重要。这一机制对进一步改进GCaMP6m和其他类似设计的生物传感器具有重要意义。
The goal of this work is to determine how GCaMP6m’s fluorescence is altered in response to Ca2+-binding. Our detailed spectroscopic study reveals the simplest explanation for how GCaMP6m changes fluorescence in response to Ca2+ is with a four-state model, in which a Ca2+-dependent change of the chromophore protonation state, due to a shift in pKa, is the predominant factor. The pKa shift is quantitatively explained by a change in electrostatic potential around the chromophore due to the conformational changes that occur in the protein when calmodulin binds Ca2+ and interacts with the M13 peptide. The absolute pKa values for the Ca2+-free and Ca2+-saturated states of GCaMP6m are critical to its high signal-to-noise ratio. This mechanism has important implications for further improvements to GCaMP6m and potentially for other similarly designed biosensors.