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

Penzkofer Penzkofer,Hegemann Hegemann,Silapetere Silapetere

doi:10.3390/ijms20174086

Penzkofer Penzkofer, Hegemann Hegemann + Show 1 more

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https://doi.org/10.3390/ijms20174086

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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 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.

Highlights

Changes in electrical potential across the plasma membrane of neurons are important for intercellular and intracellular signal transmission [1]
Two major groups of GEVIs are i) integral membrane voltage sensing domains (VSDs) composed of four trans-membrane helices fused to fluorescent proteins [15,17,18,19,20,25,26,27,29,30,31,32,33,34] and ii) microbial rhodopsins composed of 7 trans-membrane α-helices with covalently bound retinal isomers [14,16,25,26,27,35,36,37,38]
The absorption coefficient spectrum αa(λ) of a fresh thawed QuasAr1 sample was measured after centrifugation with 4400 rpm for 30 min at 4 ◦C (Centrifuge 5702 R, Eppendorf AG, Hamburg, Germany)

Summary

Introduction

Changes in electrical potential across the plasma membrane of neurons are important for intercellular and intracellular signal transmission [1]. Optical recordings of membrane potential from cells, especially neurons, with fluorescent voltage sensitive dyes [4,5,6,7,8], genetically encoded calcium indicators (GECIs) [9,10,11,12,13,14], and with fluorescent genetically encoded voltage indicators (GEVIs) [15,16,17,18,19,20,21,22,23,24,25,26,27,28] is an active field of research. Directed evolution approach yielded modified microbial rhodopsins with increased fluorescence quantum yield, and few of them exhibited change of the fluorescence intensity depending on the membrane voltage [14,15,25,26,27,35,36,37,38,42,43,44,45,46,47,48]. In rhodopsin-fluorescent protein GEVIs a microbial rhodopsin is fused with a highly fluorescent protein and the emission of the fused fluorescent protein changes upon membrane voltage changes [16,28,49,50]

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