Structural origin and rational development of bright red noncanonical variants of green fluorescent protein

Abstract

The incorporation of noncanonical amino acids (ncAAs) into fluorescent proteins is promising for red-shifting their fluorescence and benefiting tissue imaging with deep penetration and low phototoxicity. However, ncAA-based red fluorescent...

Structural Characterization of Fluorescent Proteins Using Tunable Femtosecond Stimulated Raman Spectroscopy

“…The environmental impact on the red chromophore can be seen from a recent work that reported a brighter mCherry mutant, mCherry-XL, created by mutating four residues near the RFP chromophore [110]. The mutations result in a three-fold enhancement of FQY for mCherry but blue-shifts it from 587 to 558 nm in electronic absorption, which is reminiscent of a recent report on an engineered brighter RFP with a noncanonical chromophore [95]. To discern the structural difference, the ground-state FSRS spectra under preresonance conditions were carefully compared.…”

“…For example, the most intense mode in EGFP at ~1532 cm −1 due to the quinoidal stretch (Figures 4 and 6b) is retained in its vibrational motions but blue-shifted in mPapaya1 (1558 cm −1 ), mKO2 (1558 cm −1 ), mOrange2 (1556 cm −1 ), KFP1 (1563 cm −1 ), and mCherry (1561 cm −1 , Figure 8b). The rather large blueshift (~20-30 cm −1 ) may not be solely explainable by the environmental effect [24,28,95]. Instead, it is likely caused by the EWGs (-C=N, -C=O, or -C=N-C=O) at the I-ring end that can induce a different electron density on the quinoidal stretching bonds (Figure 4).…”

“…In particular, the absence of a peak doublet between ~600 and 700 cm −1 as well as the altered intensity ratio of the ~1560 cm −1 mode over 1150 cm −1 mode in KFP1 are conspicuous (Figure 8b). It seems that the vibrational frequencies are significantly related to the structure of conjugated groups, largely regardless of the nonconjugated saturated moieties [95]. For rigorous analysis, the conformational effect due to the trans chromophore in KFP1 needs to be taken into consideration (see below).…”

“…TagRFP can also serve as a useful sample for comparison to mCherry with a cis chromophore (identical I-ring R 2 sidechain) to shed essential light on the difference between the cis and trans RFP chromophores. The corresponding model chromophores were not studied because they are not synthesizable outside the protein matrix [95].…”

“…However, we note that the calculated Raman spectra of the model chromophores are overall in poor agreement with the experimental spectra of the two RFPs (Figure 12c,d). The simplified treatment of the model chromophore in water cannot sufficiently account for the complex chromophore-environment interactions inside the protein matrix, especially for a conjugated and extended sidechain at the I-ring end that essentially increases the interplay between the chromophore (larger in size) and its surroundings (with both electrostatic and steric effects) [5,15,19,26,88,93,95,112]. In other words, although TagRFP and mCherry demonstrate substantial difference in Raman modes, the lack of a complete understanding of the environmental effects makes it inconclusive to attribute the vibrational mode difference solely to the chromophore conformation.…”