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.

Directed evolution of a far-red fluorescent rhodopsin

“…The laboratory-evolved Arch mutants described here have large Stokes shifts (differences between excitation and emission maxima) of >100 nm. Typical GFP-like proteins have very small Stokes shifts of 10-45 nm due to the rigidity of the chromophore within the protein matrix (23). The increased Stokes shift of Arch variants may be related to greater flexibility of the retinal chromophore within the protein structure.…”

“…For the single-site-saturation libraries, a mix of three mutagenic primers containing the triplet sequences NDT, VHG, and TGG were used in ratio of 12:9:1 for mutagenic PCR amplification (Table S1, primers [7][8][9][10][11][12][13][14][15][16][17][18][19][20]. For the double-site-saturation library, a mixture of 9 mutagenic primers (and a reverse primer) was used for PCR amplification (Table S1, primers [21][22][23][24][25][26][27][28][29][30]. This library design is referred to as the 22c-trick method (39).…”

“…Currently used fluorescent protein markers are based almost exclusively on Aequorea victoria GFP-like superfamily members, the most red-shifted of which emits maximally at 675 nm (23). Bacterial phytochrome receptors have recently emerged as potential substrates for the development of fluorescent markers in the NIR range (4,13,25).…”

Red fluorescent proteins (RFPs) and RFP-based biosensors for neuronal imaging applications

“…To date, the most red-shifted emission maximum for an RFP is 675 nm for the mKate variant TagRFP675. 42 …”

Blue protein with red fluorescence

“…In bacterial phytochromes, it has been proposed that proton transfer and hydrogen bond interactions play a significant role in determining their fluorescence quantum yield (28). Excited-state proton transfer (ESPT) in GFP (40)(41)(42)(43) and its variant proteins has been known for decades, which is critical in the observed redshift in their fluorescence emission (12,14,44).…”