Deciphering the molecular mechanism responsible for GCaMP6m's Ca2+-dependent change in fluorescence

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.本研究旨在明确GCaMP6m的荧光如何响应钙离子结合而发生变化。我们开展的详细光谱学研究揭示，解释GCaMP6m响应钙离子产生荧光变化的最简模型为四态模型：其中，因酸解离常数（pKa）偏移引发的生色团（chromophore）质子化状态的钙离子依赖性变化，是最为核心的影响因素。当钙调蛋白（calmodulin）结合钙离子并与M13肽段相互作用时，蛋白质会发生构象改变，进而使生色团周围的静电势发生变化，该变化可定量解释上述pKa偏移现象。GCaMP6m的无钙与钙饱和状态下的绝对pKa值，是其实现高信噪比（signal-to-noise ratio）的关键所在。该机制对于GCaMP6m的后续优化，以及其他采用同类设计的生物传感器（biosensors）的开发，均具有重要的指导意义。