Deciphering the molecular mechanism responsible for GCaMP6m's Ca2+-dependent change in fluorescence

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.

Fig 1. Schematic of Ca²⁺-saturated GCaMP6m structure.

M13 domain (orange); circularly-permuted GFP domain (green); calmodulin domain (purple). Made using 3WLD.pdb [9].

Fig 2. The Ca²⁺-dependent change in GCaMP6m fluorescence is more complicated than a chromophore transition from dim to bright.

A) Transient changes in Ca²⁺ produce different responses in the 410 nm and 480 nm excited fluorescence of GCaMP6m. This graph shows GCaMP6m response to the release of intracellular Ca²⁺ stores in HEK293 cells (average of 12 cells, mean ± standard error). Green trace represents 480 nm excited fluorescence (left axis), Blue trace represents 410 nm excited fluorescence (right axis), fluorescence emission was collected at ≥ 515 nm. Muscarinic receptor (M1) activation was used to trigger the Ca²⁺ transient. Inset image: two HEK293 cells at peak value of 480 nm excited fluorescence. B and C) show that GCaMP6m exists in an equilibrium between at least two forms of the chromophore. Absorption spectra (black trace, left axis) and excitation spectra (red trace, right axis), and emission spectra for 400 nm excited fluorescence (blue trace, right axis) and 470 nm excited fluorescence (green trace, right axis) for purified GCaMP6m protein in 0 μM free Ca²⁺ buffer (Ca²⁺-free, B) and 39 μM free Ca²⁺ buffer (Ca²⁺-saturated, C). Fluorescence emission collected at 550 nm for excitation spectra.

Fig 3. The GCaMP6m Ca²⁺-dependent spectra are consistent with four fluorescent states of the chromophore.

A and B) illustrate the Ca²⁺ concentration dependent fluorescence of GCaMP6m. 470nm (top, A) and 400 nm (top, B) excited fluorescence spectra for purified protein at 11 different buffered free Ca²⁺ concentrations, ranging from 0 μM (dark blue trace) to 39 μM (dark red trace), at pH 7.2. The 400 nm excited spectra for 39 μM free Ca²⁺ sample is marked with black arrow (B). Extended lower graphs highlight Ca²⁺ concentration dependent λ_max with normalized 470 nm (A) and 400 nm (B) excited fluorescence (same spectra as top graphs). Arrows mark the apparent peak wavelength shift between 0 μM (dark blue trace) to 39 μM (dark red trace) buffered free Ca²⁺ concentrations. C and D) Peak fluorescence intensity values from spectra in 2A and 2B, respectively, plotted against buffered free Ca²⁺ concentration. F-F_min (y-axis, 2C) is used for 470 nm excited fluorescence because baseline fluorescence in the 0 μM free Ca²⁺ buffer is the minimum fluorescence at this excitation wavelength. Alternatively, F-F_max (y-axis, 2D) is used for 400 nm excited fluorescence because baseline fluorescence in the 0 μM free Ca²⁺ buffer is the maximum fluorescence at this excitation wavelength.

Fig 4. A four state model requires the quantitative characterization of each of the four distinct Ca²⁺-dependent states of GCaMP6m.

This model illustrates a simple four state equilibrium that includes all possible combinations of the Ca²⁺-saturated and Ca²⁺-free states of the sensor protein, as well as anionic and neutral forms of the chromophore. Indices: Anionic form (A); Neutral (N); Ca²⁺-free (0); Ca²⁺-saturated (+); Quantum yield (φ); extinction coefficient (ε); Gibbs free energy of the protein (G); dissociation constant for calmodulin in GCaMP6m with anionic chromophore (K_d^(A)); dissociation for calmodulin in GCaMP6m with neutral chromophore (K_d^(N)); Acid dissociation constant for chromophore in Ca²⁺-saturated state of GCaMP6m (K_a⁽⁺⁾); Acid dissociation constant for chromophore in Ca²⁺-free state of GCaMP6m (K_a⁽⁰⁾).

Fig 5. Alkaline titration makes it possible to measure the extinction coefficient of two different forms in the same sample.

A and B) Absorption spectra for alkaline titration of purified GCaMP6m protein in 0 μM free Ca²⁺ buffer (Ca²⁺-free, A) or 39 μM free Ca²⁺ buffer (Ca²⁺-saturated, B). Individual traces represent absorption spectra at different pH values. The final two colored traces and the first black trace for Ca²⁺-free sample (A) are marked with arrows. The last colored trace and first two black traces for Ca²⁺-saturated sample are marked with arrows (B). This boundary marks a significant pH dependent change in the shape of the absorption peak near 500 nm and the last spectrum where we see linear intercorrelation of the change in OD for the two peaks. Light grey arrows below traces mark the isosbestic points. The peak value at 447 nm (black trace), gives the total concentration of chromophore. Inset plots: Absorbance values for 403 nm versus 504 nm taken from the first 8 colored traces (from pH 7.15 to 9) of the absorption spectra for Ca²⁺-free GCaMP6m (A), and for 397 nm versus 497 nm taken from all of the colored traces (from pH 7.15 to 8.29) for Ca²⁺-saturated GCaMP6m (B). These plots illustrate the change in OD for the neutral form (397 nm or 403 nm) relative to the anionic (497 nm or 504 nm) form for the respective pH ranges. The red linear fit of the first 4 data points (from pH 7.15 to 7.90) for Ca²⁺-free (A) and all 5 data points for Ca²⁺-saturated (B) in the change in OD plots gives us the ratio of the extinction coefficients for the neutral and anionic forms in the given sample.

Fig 6. GCaMP6m has a single pKa for the Ca²⁺-saturated state and two pK_a's for the Ca²⁺-free state.

Normalized 470 nm excited fluorescence intensity at 515 nm plotted against pH for Ca²⁺-saturated (solid circles) and Ca²⁺-free (open circles) states. A single-binding site model fit (green trace) was used for the Ca²⁺-saturated state and includes data points for one titration event from pH 4.3 to 8.39 with a pK_a of 7.10 ± 0.01. A two-binding site model fit (red trace) was used for the Ca²⁺-free state and includes data points for two titration events from pH 4.3 to 10.5, the first with a pK_a of 8.01 ± 0.01, and the second with a pK_a of 10.08 ± 0.02.

Fig 7. Atom numbering for the GCaMP chromophore.

Fig 8. Two-photon excitation spectra for GCaMP6m.

Spectra are presented as a linear combination of the two individual two-photon action cross-section spectra: F₂ (λ) = σ₂,_N (λ) φ_N ρ_N + σ₂,_A (λ) φ_A ρ_A, where σ₂ (λ) is the wavelength-dependent two-photon excitation cross-section, φ is the fluorescence quantum yield, and ρ is the relative concentration of the chromophore species (neutral, N, or anionic, A). Ca²⁺-saturated (solid circles) and Ca²⁺-free (open circles).