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

URN to cite this document:

urn:nbn:de:bvb:355-epub-407169 Copy

DOI to cite this document:

10.5283/epub.40716 Copy

Penzkofer, Alfons ; Silapetere, Arita ; Hegemann, Peter

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Date of publication of this fulltext: 18 Sep 2019 09:59

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Details

Item type:	Article
Journal or Publication Title:	International Journal of Molecular Sciences
Publisher:	MDPI
Volume:	20
Number of Issue or Book Chapter:	17
Page Range:	p. 4086
Date:	21 August 2019
Institutions:	Physics > Institute of Experimental and Applied Physics > Alumni or Retired Professors > Group Alfons Penzkofer
Projects:	Open Access Publizieren (DFG)
Identification Number:	Value Type 10.3390/ijms20174086 DOI
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
Dewey Decimal Classification:	500 Science > 530 Physics
Status:	Published
Refereed:	Yes, this version has been refereed
Created at the University of Regensburg:	Yes
Item ID:	40716

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Abstract

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 ...

Abstract

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

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