Abstract
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.
Highlights
Genetically-encoded Ca2+ sensors based on a single fluorescent protein are important imaging tools in neuroscience
The GCaMP sensors are constructed with circularly-permuted GFPs [8] in which the original N- and C-termini of the enhanced green fluorescent protein (EGFP) are linked together with a short, 6 amino acid peptide, and new termini are created in the middle of the 7th β-strand of the 11-stranded β-barrel, creating an opening in the side of the barrel directly adjacent to the phenolate oxygen in the chromophore
This phenomenon was observed for the earlier, GCaMP-like ratiometric pericam Ca2+ sensor [12], and indicates that the mechanism of GCaMP6m is more complicated than a simple chromophore transition from a dim state to a bright state
Summary
Genetically-encoded Ca2+ sensors based on a single fluorescent protein (i.e. non-FRET based) are important imaging tools in neuroscience. The GCaMP sensors are constructed with circularly-permuted GFPs [8] in which the original N- and C-termini of the enhanced green fluorescent protein (EGFP) are linked together with a short, 6 amino acid peptide, and new termini are created in the middle of the 7th β-strand of the 11-stranded β-barrel, creating an opening in the side of the barrel directly adjacent to the phenolate oxygen in the chromophore. A portion of the Ca2+ binding protein calmodulin, and its target peptide the M13 domain, are individually fused to the new C- and N-termini of the circularlypermuted GFP, respectively (Fig 1). The fusion of these proteins at this very sensitive location couples the Ca2+-dependent calmodulin binding of M13 to large changes in the fluorescence of the circularly-permuted GFP. More is known about the GCaMP sensors than any other biosensor, but there is still no quantitative molecular
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