Absorption and Emission Spectroscopic Investigation of the Thermal Dynamics of the Archaerhodopsin 3 Based Fluorescent Voltage Sensor Archon2
Abstract
:1. Introduction
2. Results
2.1. Absorption and Emission Behavior of Fresh Thawed Archon2 Samples
2.2. Heating-Cooling Cycle of a Fresh Thawed Archon2 Sample
2.3. Temporal Development of Archon2 at Room Temperature
2.4. Temporal Development of Archon2 at Refrigerator Temperature
3. Discussion
3.1. Behavior of Fresh Thawed Archon2
3.2. Heat Denaturation of Archon2
3.3. Thermal Aging of Archon2
3.4. Comparison of Archon2 with QuasAr1
4. Materials and Methods
4.1. Sample Preparation
4.2. Spectroscopic Measurements
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
Arch | Archaerhodopsin 3 from Halorubrum sodomense |
Archon1 | Rhodopsin based on Arch with thirteen point mutations |
Archon2 | Rhodopsin based on Arch with ten point mutations |
FRET | Förster resonance energy transfer |
GECI | Genetically encoded calcium indicator |
GEVI | Genetically encoded voltage indicator |
PRSB | Protonated retinal Schiff base |
QuasAr | Quality superior to Arch |
Ret_xxx | Retinal with absorption maximum approximately at xxx nm |
RSB | Retinal Schiff base |
Trp | Tryptophan |
Tyr | Tyrosine |
VSD | Voltage sensing domain |
References
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Parameter | Archon2 | QuasAr1 |
---|---|---|
C (mol dm−3) | 2.01 × 10−5 | 2.76 × 10−5 |
Fresh Thawed Sample | ||
Dominant PRSB | Ret_586 | Ret_580 |
λa,max (nm) | 586 | 580 |
κ | 0.784 | 0.862 |
σa(λa,max) (cm2) | 1.485 × 10−16 | 1.593 × 10−16 |
σa,int (cm) | 5.75 × 10−13 | 6.1 × 10−13 |
f | 0.546 | 0.58 |
ϕF | (1.05 ± 0.15) × 10−2 | (6.5 ± 0.5) × 10−3 |
Bmax (M−1cm−1) | 408 ± 60 | 270 ± 20 |
τrad (ns) | 10.1 | 9.32 |
τF,SB (ps) | 106 | 61.5 |
Heat Denatured Sample | ||
ϑm (°C) | 63 ± 3 | 65 ± 3 |
Dominant RSB | Ret_380 | Ret_380 |
λa,max (nm) | 380 | 380 |
σa(λa,max) (cm2) | 9.08 × 10−17 | 7.4 × 10−17 |
σa,int (cm) | 5.95 × 10−13 | 5.2 × 10−13 |
f | 0.56 | 0.49 |
ϕF | 0.008 ± 0.001 | ≈0.04 |
τrad (ns) | 3.31 | 3.74 |
τF (ps) | 26.5 | 150 |
Aging at Room Temperature | ||
ϑ (°C) | 21 ± 1 | 21–25 |
tonset of light scattering (h) | ≈48 | ≈12 |
(d−1) | ≈0.14 | ≈0 |
Ground-state isomerization | no | yes |
Aging at Refrigerator Temperature | ||
ϑ (°C) | 3 ± 1 | 2.5 ± 0.5 |
tonset of light scattering (d) | >22 | ≈50 |
(d−1) | ≈0.15 | 0.054 |
Ground-state isomerization | no | small |
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Penzkofer, A.; Silapetere, A.; Hegemann, P. Absorption and Emission Spectroscopic Investigation of the Thermal Dynamics of the Archaerhodopsin 3 Based Fluorescent Voltage Sensor Archon2. Int. J. Mol. Sci. 2020, 21, 6576. https://doi.org/10.3390/ijms21186576
Penzkofer A, Silapetere A, Hegemann P. Absorption and Emission Spectroscopic Investigation of the Thermal Dynamics of the Archaerhodopsin 3 Based Fluorescent Voltage Sensor Archon2. International Journal of Molecular Sciences. 2020; 21(18):6576. https://doi.org/10.3390/ijms21186576
Chicago/Turabian StylePenzkofer, Alfons, Arita Silapetere, and Peter Hegemann. 2020. "Absorption and Emission Spectroscopic Investigation of the Thermal Dynamics of the Archaerhodopsin 3 Based Fluorescent Voltage Sensor Archon2" International Journal of Molecular Sciences 21, no. 18: 6576. https://doi.org/10.3390/ijms21186576