Extended Stokes shift in fluorescent proteins: chromophore-protein interactions in a near-infrared TagRFP675 variant

Abstract

Most GFP-like fluorescent proteins exhibit small Stokes shifts (10-45 nm) due to rigidity of the chromophore environment that excludes non-fluorescent relaxation to a ground state. An unusual near-infrared derivative of the red fluorescent protein mKate, named TagRFP675, exhibits the Stokes shift, which is 30 nm extended comparing to that of the parental protein. In physiological conditions, TagRFP675 absorbs at 598 nm and emits at 675 nm that makes it the most red-shifted protein of the GFP-like protein family. In addition, its emission maximum strongly depends on the excitation wavelength. Structures of TagRFP675 revealed the common DsRed-like chromophore, which, however, interacts with the protein matrix via an extensive network of hydrogen bonds capable of large flexibility. Based on the spectroscopic, biochemical, and structural analysis we suggest that the rearrangement of the hydrogen bond interactions between the chromophore and the protein matrix is responsible for the TagRFP675 spectral properties.

Figure 1. Interactions between the DsRed-like chromophore and its immediate environment showed to account for a bathochromic shift in the red and far-red FPs.

(A) A hydrogen bond between the N-acylimine oxygen and a water molecule or a side chain of an amino acid found in mNeptune, eqFP650, eqFP670, mRojoA, mRouge, mPlum, and mPlum/E16Q. (B) A hydrogen bond between the protonated Glu215 carboxyl group and the imidazolinone ring nitrogen found in mCherry and Strawberry. (C) A network of hydrogen bonds between the phenolate group of the chromophore and amides of Asn143 and Asn158 found in eqFP670. (D) A hydrogen bond between the phenolate groups of the chromophore and a water molecule found in GmKate and eqFP670. (E) A hydrogen bond between the p-hydroxyphenyl group of the chromophore and the glutamate carboxyl group found in LSSmKate1. (F) A π-π-stacking interaction between the phenolate group of the chromophore and the p-hydroxyphenyl group of Tyr found in mRojoA.

Figure 2. Properties of purified TagRFP675 at pH 7.4.

(A) Absorbance spectrum. (B) Fluorescence excitation (dashed line) and emission (solid line) spectra. (C) Maturation kinetics at 37°C. (D) Photobleaching kinetics of TagRFP675 (solid line), mKate2 (dashed line), TagRFP657 (dotted line), and mNeptune (dashed-dotted line). The curves were normalized to absorbance spectra and extinction coefficients of the proteins, spectrum of an arc lamp, and transmission of a photobleaching filter.

Figure 3. Near-infrared fluorescence microscopy of live HeLa cells expressing fusion proteins.

(A) Cells (from left to right) transfected with the TagRFP675-β-actin, keratin-TagRFP675, vimentin-TagRFP675, paxillin-TagRFP675, histone-2B-TagRFP675, and α-actinin-TagRFP675 are shown. (B) The histone-2B-TagRFP657 fusion protein was co-expressed with the iRFP protein targeted to mitochondria. Images of the TagRFP675 fusions (red color) were acquired using 605/40 excitation and 640LP emission filters, and images of iRFP labeled mitochnodria (green pseudocolor) were acquired using 665/40 excitation and 725/50 emission filters. Scale bars, 10 μm.

Figure 4. Molecular structures of the TagRFP675 chromophore and its immediate environments at different pH values.

(A) Subunit A, pH 8.0. The positions of the chromophore's N-acylimine group, Ser28, and Gln41 are practically identical in all subunits. (B) Subunit A, pH 8.0. (C) Subunit B, pH 8.0. (D) Subunit A, pH 4.5. (E) Subunit B, pH 4.5. Hydrogen bonds are represented as dashed green lines with the lengths indicated in angstroms, atoms are colored by atom type, and water molecules are shown as red spheres. The occupancy of the cis- and trans-chromophore was calculated during structure refinement.

Figure 5. Effect of pH on the spectral properties of TagRFP675.

(A) Absorbance spectra at pH 3.5 (brown line), pH 4.5 (red line), pH 6.0 (orange line), pH 7.5 (green line), pH 9.0 (cyan line), and pH 10.5 (blue line). (B) Equilibrium pH dependence for TagRFP675 (solid red line) and mKate2 (dashed blue line) fluorescence. (C) Fluorescence excitation (dashed line) and emission (solid line) spectra at pH 6.0 (blue line) and pH 11.0 (red line).

Figure 6. Spectral properties of TagRFP675 and its mutants at pH 7.4.

(A) Absorbance spectra of TagRFP675 (black line), TagRFP675/Q41M (red line), and TagRFP675/Q41P (blue line). (B) Fluorescence excitation (dashed line) and emission (solid line) spectra of TagRFP675 (black line), TagRFP675/Q41M (red line), and TagRFP675/Q41P (blue line). (C) Absorbance spectra of TagRFP675 (black line), TagRFP675/N143S (red line), and TagRFP675/N158K (blue line). (D) Fluorescence excitation (dashed line) and emission (solid line) spectra of TagRFP675 (black line), TagRFP675/N143S (red line), and TagRFP675/N158S (blue line).

Figure 7. Effect of temperature on fluorescence spectra of TagRFP675, its variants, and parental mKate.

Fluorescence excitation (dashed line) and emission (solid line) spectra of (A) TagRFP675, (B) TagRFP675/Q44M, (C) TagRFP675/Q44P, and (D) mKate at 298 K (red line), 196 K (cyan line), and 77 K (blue line) are shown.

Figure 8. Dependence of fluorescence maximum on excitation wavelength at 77 K and 298 K for TagRFP675, and TagRFP675/Q41M.

(A) Maximum of the emission maximum versus the excitation wavelength of TagRFP675 at 77 K (squares) and 298 K (circles) superimposed on its absorbance spectrum at 298 K (dashed line).(B) Maximum of the emission maximum versus the excitation wavelength of TagRFP675/Q41M at 77 K (squares) and 298 K (circles) superimposed on its absorbance spectrum at 298 K (dashed line).