An Enhanced Monomeric Blue Fluorescent Protein with the High Chemical Stability of the Chromophore
Key findings
Engineered mTagBFP2 (single I174A substitution) achieves 2× greater chromophore chemical stability than mTagBFP (fluorescence half-life 88h vs 54h at pH 7.4) and 22% higher brightness in purified form (extinction coefficient 50,600 vs 41,400 M⁻¹cm⁻¹). Ex/Em: 399/454 nm.
mTagBFP2 shows 1.5× higher photostability under widefield arc lamp and 1.7× under confocal (4,152s vs 2,477s). Validated across 24 fusion constructs with proper localization. Functions as superior FRET donor with 1.13-1.2× better efficiency than mTagBFP when paired with mEGFP/mEmerald acceptors.
Chromophore degradation mechanism revealed: mTagBFP converts to non-fluorescent 330nm-absorbing hydrolyzed product (19,163.83 Da) via oxidation and imidazole ring hydrolysis, likely catalyzed by Glu215. I174A mutation stabilizes Tyr64 side chain through altered hydrogen bonding network.