Deciphering the molecular mechanism responsible for GCaMP6m's Ca(2+)-dependent change in fluorescence

The goal of this work is to determine how GCaMP6m’s fluorescence is altered in response to Ca(2+)-binding. Our detailed spectroscopic study reveals the simplest explanation for how GCaMP6m changes fluorescence in response to Ca(2+) is with a four-state model, in which a Ca(2+)-dependent change of the chromophore protonation state, due to a shift in pK(a), is the predominant factor. The pK(a) 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 Ca(2+) and interacts with the M13 peptide. The absolute pK(a) values for the Ca(2+)-free and Ca(2+)-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.