Research Article: Deciphering the molecular mechanism responsible for GCaMP6m’s Ca2+-dependent change in fluorescence

Date Published: February 9, 2017

Publisher: Public Library of Science

Author(s): Lauren M. Barnett, Thomas E. Hughes, Mikhail Drobizhev, Eugene A. Permyakov.


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.

Partial Text

Genetically-encoded Ca2+ sensors based on a single fluorescent protein (i.e. non-FRET based) are important imaging tools in neuroscience. The newest generation GCaMP6 sensors (GCaMP6s, GCaMP6m, and GCaMP6f, [1]) are bright and respond to transient increases in Ca2+ within the cell with such large changes in fluorescence that they can be used to image neuronal activity in awake, behaving animals, throughout large regions of the brain [2–5] and in entire brains [6,7].

There is a significant difference in the quantum yield and extinction coefficients between the neutral and anionic form of the chromophore, but the photophysical parameters of the anionic form do not change much upon Ca2+-binding. When calmodulin binds Ca2+, it is the redistribution of the GCaMP6m population, from the neutral to the anionic form of the chromophore, that is responsible for the large change in ~470 nm excited fluorescence. The pKa value of the chromophore strongly depends on specific amino acid positions in the chromophore environment. The change of the pKa in our measurements, from 8.01 to 7.10, is consistent with the estimation of energies based on GCaMP2 and GCaMP6m crystal structures, as there are several essential charged amino acids that shift positions with respect to the chromophore when calmodulin binds Ca2+. We predict that E386, E60, E356, D304, D382, K377, R376, and E385 are the most important. The change in pKa is important, but the absolute values of pKa are important as well. Large signals will only be produced when the pKa values of the Ca2+-free and Ca2+-saturated states straddle physiological pH. This relationship is illustrated in the comparison of the ΔF/F0 and pKa values reported for various GCaMP/GCaMP-like genetically-encoded Ca2+ sensors [9–17,19,42] and other similarly designed circularly-permuted fluorescent protein biosensors [42–46].




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