Part:BBa_K4207001

mScarlet-I

mScarlet-I is a variation of synthetic RFP with a faster maturation time. This sequence is codon-optimised for E. coli.

1. Usage and Biology

Figure 1. The absorbance and fluorescence emission spectra of mScarlet (solid line), mScalet-I (dotted line), and mScarlet-H (dashed line) by Bindels et al. (2017).

mScarlet-I is a monomeric synthetic red fluorescent protein. The protein is a variant of mScarlet, which has the longest fluorescence lifetime of any RFP up to date. The I-variant has a T74I mutation, which drastically reduces its maturation time from 174 to 36 minutes. While its brightness of 56,16 is lower than the standard mScarlet, the faster maturation time leads to faster fluorescence activity and therefore more intense signal in some applications. mScarlet-I has a maximum fluorescence at 569/593 nm (Figure 1), so it is visible to the naked eye in standard lighting.

mScarlet-I is a part of mScarlet series, a collection of three synthetic red fluorescent proteins (RFP) with different features introduced by Bindels et al in 2017. Series includes bright mScarlet, fast maturating mScarlet-I, and mScarlet-H with a fast lifetime. mScarlet-I and mScarlet-H differ from mScarlet by one amino acid substitution.

Bindels et al (2017) demonstrated that mScarlet-I as a part of mScarlet series did not exhibit any incomplete maturation, cytotoxicity, or unwanted residual dimerisation. The photochromicity of the mScarlets was also found to be negligible. This enables the usage of mScarlet-I as a FRET (Förster Resonance Energy Transfer) acceptor. They report the mScarlet series to perform well in fusion constructs due to its monomeric behavior, brightness, and low pKa.

The crystal structure of mScarlets has been defined at 1.47 Å in pH 7.8 (Bindels et al, 2017). The biggest difference between the mScarlet and mCherry crystal structures is that the phenolate ring in the chromophore is almost exactly in plane in mScarlet but out of plane in mCherry. Bindels et al (2017) list this as a possible reason for the difference in quantum yields.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 31
  Illegal EcoRI site found at 79
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 31
  Illegal EcoRI site found at 79
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 31
  Illegal EcoRI site found at 79
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 31
  Illegal EcoRI site found at 79
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 31
  Illegal EcoRI site found at 79
  
• 1000
  COMPATIBLE WITH RFC[1000]

2. References

Bindels, D., Haarbosch, L., van Weeren, L., Postma, M., Wiese, K., Mastop, M., Aumonier, S., Gotthard, G., Royant, A., Hink, M., Gadella Jr, T. (2017) mScarlet: a bright monomeric red fluorescent protein for cellular imaging. Nature Methods,14. https://doi.org/10.1038/nmeth.4074