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Article

Influence of the First Chromophore-Forming Residue on Photobleaching and Oxidative Photoconversion of EGFP and EYFP

1
Department of Chemistry, University of Southern California, Los Angeles, CA 90089, USA
2
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow 117997, Russia
3
Semenov Federal Research Center for Chemical Physics, Moscow 119991, Russia
4
Moscow Institute of Physics and Technology, Dolgoprudny, Moscow 141701, Russia
5
Present address: School of Physics, Georgia Institute of Technology, Atlanta, GA 30332, USA
6
Center of Life Sciences, Skolkovo Institute of Science and Technology, Moscow 121205, Russia
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2019, 20(20), 5229; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms20205229
Received: 27 September 2019 / Revised: 17 October 2019 / Accepted: 17 October 2019 / Published: 22 October 2019
(This article belongs to the Collection Computational Studies of Biomolecules)
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. View Full-Text
Keywords: fluorescent proteins; GFP; quantum mechanics/molecular mechanics (QM/MM); atomistic calculations; photostability; fluorescence spectroscopy; light-induced oxidation; chromophore; redding; excited-state lifetime fluorescent proteins; GFP; quantum mechanics/molecular mechanics (QM/MM); atomistic calculations; photostability; fluorescence spectroscopy; light-induced oxidation; chromophore; redding; excited-state lifetime
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MDPI and ACS Style

Sen, T.; Mamontova, A.V.; Titelmayer, A.V.; Shakhov, A.M.; Astafiev, A.A.; Acharya, A.; Lukyanov, K.A.; Krylov, A.I.; Bogdanov, A.M. Influence of the First Chromophore-Forming Residue on Photobleaching and Oxidative Photoconversion of EGFP and EYFP. Int. J. Mol. Sci. 2019, 20, 5229. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms20205229

AMA Style

Sen T, Mamontova AV, Titelmayer AV, Shakhov AM, Astafiev AA, Acharya A, Lukyanov KA, Krylov AI, Bogdanov AM. Influence of the First Chromophore-Forming Residue on Photobleaching and Oxidative Photoconversion of EGFP and EYFP. International Journal of Molecular Sciences. 2019; 20(20):5229. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms20205229

Chicago/Turabian Style

Sen, Tirthendu, Anastasia V. Mamontova, Anastasia V. Titelmayer, Aleksander M. Shakhov, Artyom A. Astafiev, Atanu Acharya, Konstantin A. Lukyanov, Anna I. Krylov, and Alexey M. Bogdanov 2019. "Influence of the First Chromophore-Forming Residue on Photobleaching and Oxidative Photoconversion of EGFP and EYFP" International Journal of Molecular Sciences 20, no. 20: 5229. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms20205229

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