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.
An Enhanced Monomeric Blue Fluorescent Protein with the High Chemical Stability of the Chromophore
The brightest monomeric blue fluorescent protein, mTagBFP, degraded over time via chromophore hydrolysis. Its degradation product was solved, and an I174A substitution produced mTagBFP2. mTagBFP2 gave a 2-fold longer half-life and better FRET efficiency with mEmerald.
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Key findings
Abstract
Commonly used monomeric blue fluorescent proteins suffer from moderate brightness. The brightest of them, mTagBFP, has a notably low chemical stability over time. Prolonged incubation of mTagBFP leads to its transition from a blue fluorescent state with absorbance at 401 nm to a non-fluorescent state with absorbance at 330 nm. Here, we have determined the chemical structure of the degraded product of the blue mTagBFP-like chromophore. On the basis of mTagBFP we have developed an improved variant, named mTagBFP2. mTagBFP2 exhibits 2-fold greater chemical stability and substantially higher brightness in live cells than mTagBFP. mTagBFP2 is also 1.2-fold and 1.7-fold more photostable than mTagBFP in widefield and confocal microscopy setups, respectively. mTagBFP2 maintains all other beneficial properties of the parental mTagBFP including the high pH stability and fast chromophore formation. The enhanced photostability and chromophore chemical stability of mTagBFP2 make it a superior protein tag. mTagBFP2 performs well in the numerous protein fusions and surpasses mTagBFP as a donor in Förster resonance energy transfer with several green fluorescent protein acceptors.