Structural characterization of IrisFP, an optical highlighter undergoing multiple photo-induced transformations

Abstract

Photoactivatable fluorescent proteins (FPs) are powerful fluorescent highlighters in live cell imaging and offer perspectives for optical nanoscopy and the development of biophotonic devices. Two types of photoactivation are currently being distinguished, reversible photoswitching between fluorescent and nonfluorescent forms and irreversible photoconversion. Here, we have combined crystallography and (in crystallo) spectroscopy to characterize the Phe-173-Ser mutant of the tetrameric variant of EosFP, named IrisFP, which incorporates both types of phototransformations. In its green fluorescent state, IrisFP displays reversible photoswitching, which involves cis-trans isomerization of the chromophore. Like its parent protein EosFP, IrisFP also photoconverts irreversibly to a red-emitting state under violet light because of an extension of the conjugated pi-electron system of the chromophore, accompanied by a cleavage of the polypeptide backbone. The red form of IrisFP exhibits a second reversible photoswitching process, which may also involve cis-trans isomerization of the chromophore. Therefore, IrisFP displays altogether 3 distinct photoactivation processes. The possibility to engineer and precisely control multiple phototransformations in photoactivatable FPs offers exciting perspectives for the extension of the fluorescent protein toolkit.

Fig. 1.

Spectroscopic characterization of IrisFP. Absorption, excitation, and emission spectra scaled to equal maximum amplitudes are depicted by solid, dashed, and dotted lines, respectively. Emission spectra of green (red) IrisFP were obtained by exciting at 488 (532) nm. Excitation spectra of green (red) IrisFP were obtained by detecting at 550 (620) nm. (A) Green IrisFP. (B) Green IrisFP before (green lines) and after (gray lines) illumination with 488-nm light. (C) Red IrisFP (red lines) after photoconversion of green IrisFP (green lines) with 405-nm light. (D) Red IrisFP before (red lines) and after (gray lines) illumination with 532-m light. Spectra were recorded on solution samples in potassium phosphate buffer, pH 9. Insets show pictures of green IrisFP crystals before (B Upper) and after (B Lower) 488-nm illumination and of red IrisFP crystals before (D Upper) and after (D Lower) 532-nm illumination, respectively. Crystals are shown in bright-field mode (Insets, Left) and in fluorescence mode (Insets, Right). Detailed experimental procedures are provided in SI Text.

Fig. 2.

Changes in the chromophore environment induced by the F173S mutation in EosFP. Common structural elements of EosFP and IrisFP are represented with green carbon atoms. Residues Phe-173 and Met-159 in EosFP are represented with gray carbon atoms, whereas the mutated residue Ser-173 and residue Met-159 in IrisFP are represented with cyan carbon atoms. The 2 water molecules (W-2188 and W-2166) are represented with cyan spheres, and W-2017 is represented with a green sphere. Hydrogen bonds are shown with dashed lines and van der Waals interactions with dotted lines.

Fig. 3.

Structural changes of the chromophore pocket upon phototransformation of IrisFP. (A) Superposition of the native green state (green) and the first reversibly switched state (black) obtained upon illumination at 488 nm. (B) Superposition of the native green state (green) and the irreversibly photoconverted red state (red) obtained after illumination at 405 nm. (C) Superposition of the red state (red) with the second reversibly switched state (black) obtained upon illumination at 405 nm, followed by illumination at 532 nm. In C, a putative model of the trans state of red IrisFP is shown (black). Hydrogen bonds are shown as dotted lines. Water molecules are shown as spheres.

Fig. 4.

Photoinduced transformations in IrisFP. Structural motions induced by light are represented by curved arrows of the same color as those used to represent light illumination at specific wavelengths. CG, cis-green Iris; tG, trans-green Iris; cR, cis-red Iris; tR, trans-red Iris.