Crystal structures of the GCaMP calcium sensor protein reveal the mechanism of fluorescence signal change and aid rational design

Abstract

The genetically encoded calcium indicator (GECI) protein GCaMP2 shows promise for neural network activity imaging, but is currently limited by low signal‐to‐noise ratio. We describe X‐ray crystal structures of GCaMP2 in the calcium‐free dark state, and in two calcium‐bound bright states: a monomeric form that dominates at intracellular concentrations observed during imaging experiments and an unexpected domain‐swapped dimer with decreased fluorescence. Based on these structures, we engineered a series of GCaMP2 point mutants and characterized one mutant with significantly improved signal‐to‐noise. The mutation is located at a domain interface and its effect on sensor function could not have been predicted in the absence of structural data.

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“…Binding calcium reversibly alters the confirmation of the GFP β‐barrel leading to desolvation, and consequent deprotonation, of fluorophore. This leads to a substantial increase in fluorescence (Akerboom et al., 2009 ; Schreiter et al., 2009 ). The REE‐sensing variant of GCaMP (termed LanTERN) has been shown to sense a range of REEs at low μM K D values, which imparts a ~ 15‐fold increase in fluorescence (Jones et al., 2024 ).…”