Mechanistic insights into reversible photoactivation in proteins of the GFP family

Abstract

Light-controlled modification of the fluorescence emission properties of proteins of the GFP family is of crucial importance for many imaging applications including superresolution microscopy. Here, we have studied the reversibly photoswitchable fluorescent protein mIrisGFP using optical spectroscopy. By analyzing the pH dependence of isomerization and protonation equilibria and the isomerization kinetics, we have obtained insight into the coupling of the chromophore to the surrounding protein moiety and a better understanding of the photoswitching mechanism. A different acid-base environment of the chromophore's protonating group in its two isomeric forms, which can be inferred from the x-ray structures of IrisFP, is key to the photoswitching function and ensures that isomerization and protonation are correlated. Amino acids near the chromophore, especially Glu212, rearrange upon isomerization, and Glu212 protonation modulates the chromophore pK(a). In mIrisGFP, the cis chromophore protonates in two steps, with pK(cis) of 5.3 and 6, which is much lower than pK(trans) (>10). Based on these results, we have put forward a mechanistic scheme that explains how the combination of isomeric and acid-base properties of the chromophore in its protein environment can produce negative and positive photoswitching modes.

Figure 1

General scheme capturing reversible photoactivation of mIrisGFP and other photoswitchable FPs. It contains eight chromophore species that are potentially involved. The states are denoted by C and T for cis and trans chromophore isomers, respectively, with superscript minus sign for anionic and superscript H for neutral; an asterisk denotes the electronically excited states.

Figure 2

Structural environment of the green chromophore in IrisFP. Hydrogen bonds are shown as dotted lines. (A) Cis conformer (PDB accession code 2VVH). (B) Trans conformer (PDB accession code 2VVI).

Figure 3

pH variation of the optical absorption spectra of mIrisGFP in the dark-adapted state (cis chromophore). (A) Absorption spectra in the pH range 3.6–10.4. Arrows point in the direction of increasing pH. (B) Peak absorption of the A_C (open circles) and B_C (solid circles) bands. Solid lines show the fit of the model depicted in panel c in the pH range 3.6–8. An additional protonation step described by the Henderson-Hasselbach equation was added to fit the B_C band amplitudes at pH >8. Dotted line shows one-site protonation with pK = 5.3. Open triangles indicate pH dependence of the reaction rate coefficient, k_off(473), for off-switching of the fluorescence with 473-nm light. (C) Four-state model describing the protonation equilibrium of the chromophore, C, interacting with a titratable amino acid, X. (D) pH dependence of the peak positions of the A_C (open circles) and B_C (solid circles) absorption bands of mIrisGFP, as well as the peak position of B_C of the Glu²¹²Gln mutant (stars).

Figure 4

pH dependence of optical spectra of mIrisGFP in the dark-adapted state (cis chromophore) (A–C) and immediately after 473-nm illumination (predominantly trans chromophore) (D–F). (A) Absorption, excitation, and emission spectra (solid, dotted, and dashed lines, respectively). Excitation and emission spectra were measured with emission at 540 nm and excitation at 390 nm. (B) CD spectra. (C) pH dependence of the (normalized) peak amplitudes of the A_C (open circles) and B_C (solid circles) absorption bands and of the emission intensity (triangles). (D and E) Absorption spectra and CD spectra, respectively. Arrows indicate increasing pH. (F) Normalized peak amplitudes of the A (open circles) and B (solid circles) bands and normalized peak shift of the A band in D (solid diamonds) and E (open diamonds) and of mutant Glu²¹²Gln (stars).

Figure 5

pH dependence of the on-switching kinetics (trans-cis isomerization). (A) Thermally activated recovery of the anionic cis chromophore with rate coefficient, k_th, measured at T = 290 K (open circles) and T = 310 K (solid circles). Stars, mIrisGFP Glu²¹²Gln at T = 310 K; solid line, two-site protonation with pK_a1 = 7.8 and pK_a2 = 9.5; dotted line, one-site protonation with pK = 7.8; dashed line, one-site protonation with pK = 10.3. (B) Photoactivated trans-cis isomerization: k_on(405) for P₄₀₅ = 13 mW cm⁻². Solid line shows one-site protonation with pK = 7.8.

Figure 6

Action spectrum of cis-trans photoisomerization of mIrisGFP. The fluorescence decrease (symbols) at 520/540 nm due to photo-induced off-switching is plotted as a function of the wavelength. Also shown are the absorption (solid line), excitation (dotted line), and emission (dashed line) spectra at pH 5.3. Action spectrum and absorption amplitudes were normalized to 1 at the peak of the A_C absorption band at 390 nm; the fluorescence spectra were normalized to match the peak of the B_C absorption band.

Figure 7

Effect of illumination power and wavelength on photo-induced off-switching of mIrisGFP (cis-trans isomerization). (A) Dependence of the rate coefficient, k_off(473), on the 473-nm excitation laser power (open circles, solid line: linear fit) and residual fluorescence upon extended 473-nm laser illumination (solid circles, dotted line: fit) as a function of laser power. (B) Time dependence of the fluorescence decay upon illumination with light at three different wavelengths.