Increasing the Fluorescence Brightness of Superphotostable EGFP Mutant by Introducing Mutations That Block Chromophore Protonation

A. V. Mamontova, A. M. Shakhov, A. P. Grigoryev, K. A. Lukyanov, A. M. Bogdanov

Research output: Contribution to journalArticlepeer-review

1 Citation (Scopus)


Abstract: The design of fluorescent proteins with increased photostability is an important practical task. One of the approaches to its solution is the search for amino acid residues, which play a key role in chromophore-mediated photochemical reactions. The effect of tyrosine-145 on the photooxidation of the chromophore in the EGFP (Enhanced Green Fluorescent Protein) has been previously demonstrated. We have designed the EGFP-Y145L mutant, which has exhibited a significantly reduced efficiency of photooxidation and associated photobleaching. In this work, we are looking for ways to increase the fluorescence brightness of this mutant. For this purpose, we have introduced the S205V and E222G substitutions and their combination to shift the pH equilibrium of the chromophore environment towards ionization of the chromophore. We have shown that both mutants that contain the S205V substitution carry mostly the neutral chromophore, have low brightness, and are capable of slow photoactivation. They may be of interest for studying light-dependent proton transfer. Probably, these proteins can function as time-resolved pH sensors. As we have expected, the chromophore resides in the EGFP-Y145L/E222G mutant is predominantly in the anionic form. The fluorescence brightness of this protein is four times higher than that of the original EGFP-Y145L, and its photostability is higher than that of EGFP by factors of 1.5–5.

Original languageEnglish
Pages (from-to)1229-1241
Number of pages13
JournalRussian Journal of Bioorganic Chemistry
Issue number6
Publication statusPublished - Nov 2020


  • chromophore
  • ESPT
  • excited state lifetime
  • fluorescence spectroscopy
  • fluorescent proteins
  • GFP
  • photoinduced oxidation
  • photostability


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