Penzkofer, Alfons and Silapetere, Arita and Hegemann, Peter (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 (18): 6576. ISSN , 1422-0067
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Archon2 is a fluorescent voltage sensor derived from Archaerhodopsin 3 (Arch) of Halorubrum sodomense using robotic multidimensional directed evolution approach. Here we report absorption and emission spectroscopic studies of Archon2 in Tris buffer at pH 8. Absorption cross-section spectra, fluorescence quantum distributions, fluorescence quantum yields, and fluorescence excitation spectra were determined. The thermal stability of Archon2 was studied by long-time attenuation coefficient measurements at room temperature (21 +/- 1 degrees C) and at refrigerator temperature (3 +/- 1 degrees C). The apparent melting temperature was determined by stepwise sample heating up and cooling down (obtained apparent melting temperature: 63 +/- 3 degrees C). In the protein melting process protonated retinal Schiff base (PRSB) with absorption maximum at 586 nm converted to de-protonated retinal Schiff base (RSB) with absorption maximum at 380 nm. Storage of Archon2 at room temperature and refrigerator temperature caused absorption coefficient decrease because of partial protein clustering to aggregates at condensation nuclei and sedimentation. At room temperature an onset of light scattering was observed after two days because of the beginning of protein unfolding. During the period of observation (18 days at 21 degrees C, 22 days at 3 degrees C) no change of retinal isomer composition was observed indicating a high potential energy barrier of S-0 ground-state isomerization.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | ALL-OPTICAL ELECTROPHYSIOLOGY; F-2(-) COLOR-CENTERS; AGGREGATION; LIGHT; BEHAVIOR; RED; Archon2; Archaerhodopsin 3; genetically encoded voltage sensor (GEVI); absorption spectroscopic characterization; fluorescence spectroscopic characterization; apparent protein melting temperature; thermal stability |
| Subjects: | 500 Science > 530 Physics |
| Divisions: | Physics > Institute of Experimental and Applied Physics Physics > Institute of Experimental and Applied Physics > Alumni or Retired Professors > Group Alfons Penzkofer Physics > Institute of Experimental and Applied Physics > Alumni or Retired Professors |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 12 Mar 2021 09:08 |
| Last Modified: | 12 Mar 2021 09:08 |
| URI: | https://pred.uni-regensburg.de/id/eprint/43875 |
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