Influence of the First Chromophore-Forming Residue on Photobleaching and Oxidative Photoconversion of EGFP and EYFP
Abstract
:1. Introduction
2. Results
2.1. Mutants General Description (Spectral Characteristics)
2.2. Photostability
2.3. Redding
2.4. Lifetimes
2.5. Computational Results
- Quantum-chemical calculations of the isolated model chromophores (structures, excitation energies, and oscillator strengths);
- Molecular dynamics simulations of the model proteins in the ground and electronically excited states;
- Hybrid QM/MM (quantum mechanics/molecular mechanics) calculations of the spectral properties of the model proteins (excitation energies and oscillator strengths for the structures taken from the ground-state molecular dynamics simulations).
3. Discussion
- -
- EGFP-T65G has a larger extinction coefficient than EGFP because of its larger oscillator strength;
- -
- Because of the faster radiationless relaxation, EGFP-T65G has lower FQY than EGFP, as per Equation (5); likewise, EYFP has lower FQY than EYFP-G65T;
- -
- Having glycine in position 65 leads to faster radiationless relaxation (shorter half-life of A), thus suppressing the bleaching and leading to an increased photostability;
- -
- Larger FQY in EYFP relative to EGFP-T65G arises due to the suppression of torsional motions by the π-stacking interactions, which is reflected in longer radiationless half-life.
4. Materials and Methods
4.1. Spectroscopy and Fluorescence Brightness Evaluation
4.2. Microscopy
4.3. Protein Expression and Purification
4.4. Site-Directed Mutagenesis
4.5. Fluorescence Lifetime Imaging Microscopy of the Purified Proteins upon Single-Photon Excitation
4.6. Computational Details
4.6.1. Protonation State and Crystal Structures
4.6.2. Molecular Dynamics Setup
- Minimization for 2000 steps with 2 fs time step.
- Equilibration of the solvent (keeping the protein frozen) for 500 ps with 1 fs time step.
- Equilibration of the system for 2 ns (with 1 fs time step) with periodic boundary condition (PBC) under the isobaric–isothermal NPT ensemble.
- Production run for 2 ns with 1 fs time step in an NPT ensemble.
4.6.3. QM/MM Setup
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
Abbreviations
avGFP | Aequorea victoria green fluorescent protein |
EC | Extinction coefficient |
EGFP | Enhanced green fluorescent protein |
ET | Electron transfer |
EYFP | Enhanced yellow fluorescent protein |
FL | Fluorescence lifetime |
FP | Fluorescent protein |
FQY | Fluorescence quantum yield |
GFP | Green fluorescent protein |
GYG | Glycine-tyrosine-glycine |
PES | Potential energy surface |
PB | Phosphate buffer |
PBS | Phosphate buffered saline |
QY | Quantum yield |
SYG | Serine-tyrosine-glycine |
TYG | Threonine-tyrosine-glycine |
QM/MM | Quantum mechanics/molecular mechanics |
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Fluorescent Protein | λex/λem, nm | EC, M−1cm−1 | FQY | Relative Brightness, % * | Fluorescence Lifetime, ns ** | Photobleaching, s # | Redding Rate | |||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
PB | PBS | PB | PBS | PB + Ox | PBS + Ox | PB | PBS | |||||
EGFP | 489/509 | 55000 | 0.60 | 100 | n/d | 2.8 | n/d | 80 ± 10 | n/d | 5 ± 2.5 | n/d | strong |
EGFP-T65G | 488/508 | 70000 | 0.06 | 13 | n/d | 1.3 | n/d | 170 ± 25 | n/d | 85 ± 15 | n/d | weak |
EYFP | 514/526 | 83400 | 0.61 | 154 | 3.18 ± 0.07 | 3.0 ± 0.08 | 21 ± 2 | 35 ± 2 | 3 ± 2 | 2 ± 1 | no | moderate |
EYFP-G65T | 510/525 | n/d | 0.78 | n/d | 3.7 ± 0.06/4.0 ± 0.09 | 3.5 ± 0.07 and 0.5 ± 0.07/ 3.9 ± 0.08 | 25 ± 8 | 180 ± 8 | 10 ± 2 | 32 ± 7 | moderate | moderate |
Protein/Chro | EGFP/ TYG | EGFP-T65G/ GYG | EYFP/ GYG | EYFP-G65T/ TYG | EYFP+Cl–/ GYG |
---|---|---|---|---|---|
Average No. of hbond | 2.81 | 2.31 | 1.34 | 1.93 | 1.45 |
STD (hbond) | 1.12 | 1.03 | 0.83 | 1.06 | 0.87 |
Δ | 7.40 | 6.44 | 4.41 | 5.50 | 7.02 |
STD (Δ) | 16.7 | 8.2 | 7.8 | 8.0 | 7.29 |
Protein | Eex, eV (fl) | τfl, ns | τfl,rel | ||||
---|---|---|---|---|---|---|---|
Gas phase | QM/MM | Gas phase (n = 1) | QM/MM (n = 1) | QM/MM (n = 1.6) | Gas phase | QM/MM | |
EGFP | 3.101 (1.02) | 3.081 (0.97) | 29.50 | 31.24 | 7.63 | 1.00 | 1.00 |
EGFP-T65G | 3.123 (1.05) | 3.142 (1.04) | 28.25 | 28.18 | 6.88 | 0.95 | 0.90 |
EYFP | 3.123 (1.05) | 3.097 (1.05) | 28.25 | 28.71 | 7.02 | 0.95 | 0.92 |
EYFP-G65T | 3.101 (1.02) | 3.015 (0.98) | 29.50 | 32.49 | 7.94 | 1.00 | 1.04 |
EYFP+Cl– | 3.123 (1.05) | 3.077 (1.07) | 28.25 | 28.57 | 6.97 | 0.95 | 0.91 |
Protein | τfl, ns (τfl,rel) a | τnr, ns (τnr,rel) | τ, ns (τrel) b | FQY | Ybl, rel c |
---|---|---|---|---|---|
EGFP | 7.63 (1.00) | 5.92 (1.00) | 3.33 (1.0) | 0.44 | 1.0 |
EGFP-T65G | 6.88 (0.90) | 0.25 (0.04) | 0.24 (0.1) | 0.04 | 0.1 |
EYFP | 7.02 (0.92) | 1.73 (0.29) | 1.39 (0.4) | 0.20 | 0.4 |
EYFP-G65T | 7.94 (1.04) | 10.8 (1.82) | 4.58 (1.4) | 0.58 | 1.4 |
EYFP+Cl– | 6.97 (0.91) | 0.57 (0.10) | 0.53 (0.2) | 0.07 | 0.2 |
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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://doi.org/10.3390/ijms20205229
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://doi.org/10.3390/ijms20205229
Chicago/Turabian StyleSen, 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://doi.org/10.3390/ijms20205229