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Open AccessArticle

Absorption and Emission Spectroscopic Investigation of the Thermal Dynamics of the Archaerhodopsin 3 Based Fluorescent Voltage Sensor QuasAr1

1
Fakultät für Physik, Universität Regensburg, Universitätsstraße 31, D-93053 Regensburg, Germany
2
Experimentelle Biophysik, Institut für Biologie, Humboldt Universität zu Berlin, Invalidenstraße 42, D-10115 Berlin, Germany
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2019, 20(17), 4086; https://doi.org/10.3390/ijms20174086
Received: 21 July 2019 / Revised: 15 August 2019 / Accepted: 16 August 2019 / Published: 21 August 2019
QuasAr1 is a fluorescent voltage sensor derived from Archaerhodopsin 3 (Arch) of Halorubrum sodomense by directed evolution. Here we report absorption and emission spectroscopic studies of QuasAr1 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 QuasAr1 was studied by long-time attenuation coefficient measurements at room temperature (23 ± 2 °C) and at 2.5 ± 0.5 °C. The apparent melting temperature was determined by stepwise sample heating up and cooling down (obtained apparent melting temperature: 65 ± 3 °C). In the protein melting process the originally present protonated retinal Schiff base (PRSB) with absorption maximum at 580 nm converted to de-protonated retinal Schiff base (RSB) with absorption maximum at 380 nm. Long-time storage of QuasAr1 at temperatures around 2.5 °C and around 23 °C caused gradual protonated retinal Schiff base isomer changes to other isomer conformations, de-protonation to retinal Schiff base isomers, and apoprotein structure changes showing up in ultraviolet absorption increase. Reaction coordinate schemes are presented for the thermal protonated retinal Schiff base isomerizations and deprotonations in parallel with the dynamic apoprotein restructurings. View Full-Text
Keywords: QuasArs; Archaerhodopsin 3; genetically encoded voltage sensors (GEVIs); absorption spectroscopic characterization; fluorescence spectroscopic characterization; apparent protein melting temperature; thermal stability; thermal isomerization; thermal deprotonation QuasArs; Archaerhodopsin 3; genetically encoded voltage sensors (GEVIs); absorption spectroscopic characterization; fluorescence spectroscopic characterization; apparent protein melting temperature; thermal stability; thermal isomerization; thermal deprotonation
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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 QuasAr1. Int. J. Mol. Sci. 2019, 20, 4086.

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