Structure-guided evolution of cyan fluorescent proteins towards a quantum yield of 93%
Key findings
X-ray crystallography of SCFP3A and mTurquoise at high resolution revealed that stabilizing the seventh β‑strand and strengthening the principal chromophore hydrogen bond reduced non-radiative excited-state decay. Structural refinement showed improved packing of the chromophore environment, explaining increased quantum yield and lifetime.
Site-saturation mutagenesis at residue 146 combined with fluorescence lifetime–based screening identified the I146F substitution that produced mTurquoise2. Purified protein measurements gave a quantum yield of 0.93 (≈10% higher than mTurquoise) and an extinction coefficient of 30,000 M⁻1cm⁻1, yielding the highest reported QY for a monomeric fluorescent protein and increased molecular brightness.
In mammalian-cell characterization mTurquoise2 matured faster and delivered almost 20% higher cellular brightness versus mTurquoise, showed high photostability, and displayed a mono-exponential fluorescence lifetime near 4.0 ns. The calculated Förster radius for the mTurquoise2–mVenus pair was 58.3 Å (≈1 Å larger than mTurquoise–mVenus), improving quantitative FRET-FLIM performance.