Engineering ESPT pathways based on structural analysis of LSSmKate red fluorescent proteins with large Stokes shift

Abstract

LSSmKate1 and LSSmKate2 are monomeric red fluorescent proteins (RFPs) with large Stokes shifts (LSSs), which allows for efficient separation of absorbance and emission maxima, as well as for excitation with conventional two-photon laser sources. These LSSmKates differ by a single amino acid substitution at position 160 and exhibit absorbance maxima around 460 nm, corresponding to a neutral DsRed-like chromophore. However, excitation at 460 nm leads to fluorescence emission above 600 nm. Structures of LSSmKate1 and LSSmKate2, determined at resolutions of 2.0 and 1.5 A, respectively, revealed that the predominant DsRed-chromophore configurations are cis for LSSmKate1 but trans for LSSmKate2. Crystallographic and mutagenesis analyses, as well as isotope and temperature dependences, suggest that an excited-state proton transfer (ESPT) is responsible for the LSSs observed in LSSmKates. Hydrogen bonding between the chromophore hydroxyl and Glu160 in LSSmKate1 and a proton relay involving the chromophore tyrosine hydroxyl, Ser158, and the Asp160 carboxylate in LSSmKate2 represent the putative ESPT pathways. Comparisons with mKeima LSS RFP suggest that similar proton relays could be engineered in other FPs. Accordingly, we mutated positions 158 and 160 in several conventional red-shifted FPs, including mNeptune, mCherry, mStrawberry, mOrange, and mKO, and the resulting FP variants exhibited LSS fluorescence emission in a wide range of wavelengths from 560 to 640 nm. These data suggest that different chromophores formed by distinct tripeptides in different environments can be rationally modified to yield RFPs with novel photochemical properties.

Figure 1

Amino acid sequence alignment of LSSmKate1 and LSSmKate2 with mKeima, mKate, mNeptune, mCherry, mStrawberry, mOrange, and mKO. Residues facing the interior of the protein’s β-barrel are highlighted in light gray; α-helices are shown as ribbons; β-strands are shown as arrows. The chromophore-forming residues are underlined. The proton acceptor amino acid residues in LSSmKate1, LSSmKate2, and mKeima are shown in white on black. Mutated amino acids are shown in white italic on dark gray. The alignment numbering follows that of LSSmKate proteins.

Figure 2

Molecular structures of the LSSmKate1 (A) and LSSmKate2 (B) chromophores and their immediate environments, superimposed over the corresponding 2Fo-Fc electron density (contoured at 1 σ). Hydrogen bonds are represented as dashed green lines with the lengths indicated in Å; atoms are colored by atom type; water molecules are shown as red spheres. The arrows indicate χ₁ and χ₂ torsion angles.

Figure 3

Absorbance and fluorescence spectra of LSSmKate1 (A, C) and LSSmKate2 (B, D) at different pH values (see also Table 1 and Supporting Figure 2). In panels C and D, the absorbance spectra at pH 7 were measured after the equilibration of protein samples from pH 11 back to the neutral pH value.

Figure 4

Fluorescence emission spectra of LSSmKate1 (A) and LSSmKate2 (B) in two different solvents at 77K and 298K are shown. The protein samples were excited at 450 nm. The spectra are normalized to 100%.

Figure 5

Proton relays of LSSmKate1 (A), LSSmKate2 (B), and mKeima (C) are shown for the chromophore and the residues involved in ESPT (hydrogen bonds are highlighted in gray).

Figure 6

Fluorescence excitation and emission spectra are shown for mKate/158D (solid line) and mKate (dotted line) (A); mKate/158G/160E (solid line) and mKate (dotted line) (B); mNeptune/158D (solid line) and mNeptune (dotted line) (C); mNeptune/158S/160E (solid line) and mNeptune (dotted line); mCherry/158E/160A (solid line) and mCherry (dotted line) (E); mCherry/158S/160E (solid line) and mCherry (dotted line) (F); mOrange 158D/160L (solid line) and mOrange (dotted line) (G); mOrange/158A/160E (solid line) and mOrange (dotted line) (H); mStrawberry/158D/160L (solid line) and mStrawberry (dotted line) (I); mKO/158E (solid line) and mKO (dotted line) (J).

Figure 7

Proposed photocycles for LSSmKate1 (panel A) and LSSmKate2 (panel B) at neutral pH and room temperature are shown. The excited state (compound A*) of the neutral chromophore, which is photo-excited from the ground state (compound A), is transformed after ESPT into the intermediate excited state denoted as the compound I*. After fluorescence emission, proton transfer from Glu160 (LSSmKate1) or Asp160 (LSSmKate2) to the chromophore regenerates the ground state (compound A) of the neutral chromophore.