Influence of the first chromophore-forming residue on photobleaching and oxidative photoconversion of EGFP and EYFP

Tirthendu Sen, Anastasia V. Mamontova, Anastasia V. Titelmayer, Aleksander M. Shakhov, Artyom A. Astafiev, Atanu Acharya, Konstantin A. Lukyanov, Anna I. Krylov, Alexey M. Bogdanov

Research output: Contribution to journalArticlepeer-review

12 Scopus citations


Enhanced green fluorescent protein (EGFP)—one of the most widely applied genetically encoded fluorescent probes—carries the threonine-tyrosine-glycine (TYG) chromophore. EGFP efficiently undergoes green-to-red oxidative photoconversion (“redding”) with electron acceptors. Enhanced yellow fluorescent protein (EYFP), a close EGFP homologue (five amino acid substitutions), has a glycine-tyrosine-glycine (GYG) chromophore and is much less susceptible to redding, requiring halide ions in addition to the oxidants. In this contribution we aim to clarify the role of the first chromophore-forming amino acid in photoinduced behavior of these fluorescent proteins. To that end, we compared photobleaching and redding kinetics of EGFP, EYFP, and their mutants with reciprocally substituted chromophore residues, EGFP-T65G and EYFP-G65T. Measurements showed that T65G mutation significantly increases EGFP photostability and inhibits its excited-state oxidation efficiency. Remarkably, while EYFP-G65T demonstrated highly increased spectral sensitivity to chloride, it is also able to undergo redding chloride-independently. Atomistic calculations reveal that the GYG chromophore has an increased flexibility, which facilitates radiationless relaxation leading to the reduced fluorescence quantum yield in the T65G mutant. The GYG chromophore also has larger oscillator strength as compared to TYG, which leads to a shorter radiative lifetime (i.e., a faster rate of fluorescence). The faster fluorescence rate partially compensates for the loss of quantum efficiency due to radiationless relaxation. The shorter excited-state lifetime of the GYG chromophore is responsible for its increased photostability and resistance to redding. In EYFP and EYFP-G65T, the chromophore is stabilized by π-stacking with Tyr203, which suppresses its twisting motions relative to EGFP.

Original languageEnglish (US)
Article number5229
JournalInternational journal of molecular sciences
Issue number20
StatePublished - Oct 2019
Externally publishedYes


  • Atomistic calculations
  • Chromophore
  • Excited-state lifetime
  • Fluorescence spectroscopy
  • Fluorescent proteins
  • GFP
  • Light-induced oxidation
  • Photostability
  • Quantum mechanics/molecular mechanics (QM/MM)
  • Redding

ASJC Scopus subject areas

  • Catalysis
  • Molecular Biology
  • Spectroscopy
  • Computer Science Applications
  • Physical and Theoretical Chemistry
  • Organic Chemistry
  • Inorganic Chemistry


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