Directed evolution of a monomeric, bright and photostable version of Clavularia cyan fluorescent protein: structural characterization and applications in fluorescence imaging

Abstract

The arsenal of engineered variants of the GFP [green FP (fluorescent protein)] from Aequorea jellyfish provides researchers with a powerful set of tools for use in biochemical and cell biology research. The recent discovery of diverse FPs in Anthozoa coral species has provided protein engineers with an abundance of alternative progenitor FPs from which improved variants that complement or supersede existing Aequorea GFP variants could be derived. Here, we report the engineering of the first monomeric version of the tetrameric CFP (cyan FP) cFP484 from Clavularia coral. Starting from a designed synthetic gene library with mammalian codon preferences, we identified dimeric cFP484 variants with fluorescent brightness significantly greater than the wild-type protein. Following incorporation of dimer-breaking mutations and extensive directed evolution with selection for blue-shifted emission, high fluorescent brightness and photostability, we arrived at an optimized variant that we have named mTFP1 [monomeric TFP1 (teal FP 1)]. The new mTFP1 is one of the brightest and most photostable FPs reported to date. In addition, the fluorescence is insensitive to physiologically relevant pH changes and the fluorescence lifetime decay is best fitted as a single exponential. The 1.19 A crystal structure (1 A=0.1 nm) of mTFP1 confirms the monomeric structure and reveals an unusually distorted chromophore conformation. As we experimentally demonstrate, the high quantum yield of mTFP1 (0.85) makes it particularly suitable as a replacement for ECFP (enhanced CFP) or Cerulean as a FRET (fluorescence resonance energy transfer) donor to either a yellow or orange FP acceptor.

Quantitative imaging of endosome acidification and single retrovirus fusion with distinct pools of early endosomes

“…Low pH markedly reduces sensitized emission of eYFP (pK a 7.1) (20) by shifting its absorption peak (21) (Fig. 1 B and C) and thereby increases the signal from relatively pH-insensitive mTFP1 (pK a 4.3) (17).…”

“…The CFP (donor) module of pHlameleon was replaced with the monomeric teal fluorescent protein, mTFP1 (Fig. 1A) (17). The higher photostability and brightness of mTFP1 compared with CFP make this protein more suitable for single-virus imaging.…”

“…Normally, FRET measurements using sensitized emission require corrections for direct excitation of the acceptor by the 458-nm laser line and spectral bleed-through (donor photons detected in the acceptor channel). However, for intramolecular FRET between the tandem mTFP1-eYFP proteins, the donor/acceptor stoichiometry and their emission ratio are fixed, thus providing a reliable direct measure of pH based on the emission ratio (17).…”

Extracellular matrix structure governs invasion resistance in bacterial biofilms

“…All strains harbored a chromosomal copy of the fluorescent protein mTFP1 (Ai et al, 2006), mKO (Karasawa et al, 2004) or mKate (Shcherbo et al, 2007). In all cases, fluorescent proteins were expressed constitutively.…”

Future Perspective of Single-Molecule FRET Biosensors and Intravital FRET Microscopy

“…Indeed, by replacing CFP with teal FP, an FP derived from Clavularia sp. (coral) (25) with less homology to CFP, we established stable cell lines expressing FRET biosensors for small GTPases and phosphoinositides and observed their activity and concentration in organoids (26)(27)(28). Also, many laboratories have utilized a GFP-red-FP (RFP) pair, which originates from Aequorea victoria (jellyfish) and Discosoma sp.…”