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

  • ️Thu Jan 01 2009

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

Jasper Akerboom et al. J Biol Chem. 2009.

Abstract

The genetically encoded calcium indicator GCaMP2 shows promise for neural network activity imaging, but is currently limited by low signal-to-noise ratio. We describe x-ray crystal structures as well as solution biophysical and spectroscopic characterization 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. This series of structures provides insight into the mechanism of Ca2+-induced fluorescence change. Upon calcium binding, the calmodulin (CaM) domain wraps around the M13 peptide, creating a new domain interface between CaM and the circularly permuted enhanced green fluorescent protein domain. Residues from CaM alter the chemical environment of the circularly permuted enhanced green fluorescent protein chromophore and, together with flexible inter-domain linkers, block solvent access to the chromophore. Guided by the crystal structures, we engineered a series of GCaMP2 point mutants to probe the mechanism of GCaMP2 function 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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Figures

FIGURE 1.
FIGURE 1.

Crystal structures of GCaMP2. A, schematic of the primary amino acid sequence of GCaMP2 illustrating the domain organization. Domains are colored as depicted in B-D. Carets below the schematic show the positions of inter-domain linkers whose amino acid sequences are given. B, stereoview of the structure of the Ca2+-saturated domain-swapped GCaMP2 dimer, depicted as ribbons. One molecule of the dimer is colored by domain as in A, the other molecule is colored light gray. The EGFP chromophore is represented as sticks and calcium ions are shown as orange spheres. C, structure of Ca2+-saturated GCaMP2 monomer, represented as in B except the domains are labeled. D, structure of calcium-free GCaMP2, represented as in B and C. Note that the M13 peptide and the C-terminal half of CaM are not included in the model due to lack of electron density, suggesting their flexibility. This and other structure figures were prepared using PyMOL (Delano Scientific, San Carlos, CA).

FIGURE 2.
FIGURE 2.

Solution studies of monomeric and dimeric GCaMP2. A, SEC of calcium-free (red) and calcium-bound (blue) GCaMP2 following affinity purification. Peaks are labeled with the assigned species and estimated molecular mass. The standard curve used to estimate Mr is in the inset. The predicted molecular mass for GCaMP2 monomer and dimer are 50.7 and 101.3 kDa, respectively. B, c(s) distribution analysis of sedimentation velocity analytical ultracentrifugation data for calcium-free (red) and calcium-bound (blue) GCaMP2 following affinity purification. Peaks are labeled as in B. C, SEC analysis of a nearly equimolar mixture of purified, Ca2+-saturated GCaMP2 monomer and dimer, monitoring protein absorbance at 280 nm (black), cpEGFP chromophore absorbance at 495 nm (blue), and cpEGFP fluorescence at 488/510 nm (red). Note the difference in relative chromophore absorbance and fluorescence of the dimer. D, fluorescence excitation (solid lines) and emission (dashed lines) spectra of isolated Ca2+-saturated GCaMP2 monomer (blue) and dimer (red), as well as calcium-free GCaMP2 (black). Absorbance spectra of these three species are shown in the inset.

FIGURE 3.
FIGURE 3.

Stereoview of the structures of Ca2+-saturated GCaMP2-K387W monomer (red), Ca2+-saturated GCaMP2 dimer (yellow), and Ca2+-free 8EF-GCaMP2 (blue) superimposed using the cpEGFP domain. The proteins are represented as ribbons with the cpEGFP chromophore represented as sticks.

FIGURE 4.
FIGURE 4.

Fluorescent properties of GCaMP2 variants. A, ΔF/F0 for calcium binding of all GCaMP2 mutants listed in supplemental Table S3. All GCaMP2 mutants listed also contain the T116V mutation. B, normalized fluorescence of Ca2+-free (apo, white) and the Ca2+-saturated (Sat, gray) state of the GCaMP2 variants. Fluorescence of both states is normalized to the brightness of GCaMP2 T116V. Note that although GCaMP2 T116V-T303Y has a greater ΔF/F0 (panel A), the brightness of both Ca2+-free and Ca2+-saturated state is lower than for GCaMP2 T116V.

FIGURE 5.
FIGURE 5.

A rationally designed, improved GCaMP2 variant. A, fluorescence excitation (solid lines) and emission (dashed lines) spectra of Ca2+-saturated GCaMP2 T116V (red) and T116V/D381Y (blue), as well as their calcium-free forms (gray and black, respectively). Normalized absorbance spectra of each are shown in the inset. B, close-up stereo view of the Ca2+-saturated monomeric GCaMP2 structure, showing the location of aspartate 381 (D381) of CaM at the CaM/cpEGFP domain interface. GCaMP2 is displayed as ribbons colored by domain. The side chain of Asp381 and the cpEGFP chromophore are displayed as sticks. A model of a tyrosine side chain at position 381 is shown in semitransparent sticks to represent a possible conformation of the D381Y mutant and to illustrate the proximity of this side chain to the chromophore.

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