Part:BBa_K5316013

SVFP

Sirius Violet Fluorescent Protein is a pH-insensitive and photostable protein with an emission wavelength of 424 nm. It allows prolonged visualization of biological events in an acidic environment.

For several years, the Phe66 variant of wild-type Aequorea victoria GFP had the shortest emission wavelength, peaking at 442 nm. However, its low fluorescence quantum yield (ΦFl = 0.013) significantly limited its use in bioimaging applications.

To improve the brightness of the GFP-Phe66 variant, Tomosugi et al. conducted random mutagenesis on the region encoding four amino acids surrounding the chromophore of mseCFP-W66F, a cyan fluorescent protein variant developed by introducing the W66F mutation into mseCFP. One of the resulting mutants, with four substitutions (T65Q, Y145G, H148S, and T203V), showed a 25-fold increase in fluorescence intensity and was classified as an ultramarine fluorescent protein.

This variant, named UMFP-1 or Sirius, has an emission peak at 424 nm and two absorption peaks at 280 and 355 nm. Due to its strongly blue-shifted spectrum, Sirius is spectrally compatible for multicolor imaging with CFP, YFP, and RFP, and can be used in a separate detection channel from BFP, enabling extended multicolor imaging through linear spectral unmixing.

Sirius was considered by Tomosugi et al. a promising donor for pairing with mseCFP in fluorescence resonance energy transfer (FRET), as its emission spectrum significantly overlaps with the absorption spectrum of mseCFP.

However, the degree of spectral overlap (J = 0.71 × 10−13 M−1 cm3) is smaller than in other common FRET pairs like CFP-YFP (J = 1.89 × 10−13 M−1 cm3), due to the wavelength dependency of J.

Moreover, with an assumed κ² value of 2/3, the Förster distance (R0) for the Sirius-mseCFP pair (3.7 nm) is shorter than that of CFP-YFP (4.7 nm), owing to the lower fluorescence quantum yield of Sirius.

Despite these drawbacks, the Sirius-mseCFP fusion protein, linked via a Leu-Glu peptide, exhibits moderate sensitized emission from mseCFP, with a FRET efficiency of 43%. Building on this result, Tomosugi et al. develop an indicator for caspase-3 activation by fusing the caspase-3 substrate Asp-Glu-Val-Asp between Sirius and mseCFP, creating SC-SCAT3. They observe changes in the FRET signal of SC-SCAT3 in apoptotic HeLa cells, detecting caspase-3 activation first in the cytoplasm and then in the nucleus, followed by cell shrinkage.

(All of the information provided in this description has been adapted from Tomosugi et al.’s paper: “An ultramarine fluorescent protein with increased photostability and pH insensitivity”).

Design considerations

The iGEM Barcelona-UB team of 2024 reverse-translated the original protein sequence from Tomosugi et al. to match the most frequently used codons in Homo sapiens, improving its compatibility with human cells for better expression.

Reverse transcription was conducted using the Sequence Manipulation Suite from bioinformatics.org (https://www.bioinformatics.org/sms2/rev_trans.html), using the codon frequencies provided by Kazusa (https://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=9606&aa=1&style=GCG)

References

Sequence sourced from Nat Methods. 2009 May;6(5):351-3: https://pubmed.ncbi.nlm.nih.gov/19349978/