Absorption and Emission Spectroscopic Investigation of the Thermal Dynamics of the Archaerhodopsin 3 Based Fluorescent Voltage Sensor Archon2

Abstract

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 °C) and at refrigerator temperature (3 ± 1 °C). The apparent melting temperature was determined by stepwise sample heating up and cooling down (obtained apparent melting temperature: 63 ± 3 °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 °C, 22 days at 3 °C) no change of retinal isomer composition was observed indicating a high potential energy barrier of S0 ground-state isomerization.

Fluorescence of the Retinal Chromophore in Microbial and Animal Rhodopsins

“…Table 1 contains the fluorescence excitation maxima (λ exc ), maxima of fluorescence band (λ em ), quantum yields of fluorescence (Φ), and excited state lifetimes (τ f ) that are reported for wild-type microbial rhodopsins and their mutants measured in a wide range of pH values, as well as for the O and Q photocycle intermediates of bacteriorhodopsin. 0.5 [26] 0.56 [30] bR blue 610-620 [31] 613-614 [31] 750 [29] 130-140 4.5 × 10 −3 [31] 1.5, 8.6 [32] 7.8 [30] bR O 635-640 [33] -750 ± 5 [28] 110-120 Arch 553 [24] 553 [24] 687 [24] 134 1.1 × 10 −4 [24] 9 × 10 −4 [37] 5.9, 60 (intense illumination) [24] [23,38] 590 [39] 585 [38] 715 [39] 740 [38] 135, 160 8 × 10 −3 [39] 6.5 × 10 −3 [38] 61.5 [38] Archon2 586 [40] -735 [40] 149 1.05 × 10 −2 [40] 106 [40] Archer1 626 [41] 627 [42] 731 [41] 103 3.3 × 10 −3 [41] -PR 531 [13] 565 [13] 700 [13] 169 2 × 10 −4 [13] 1.4 [13] 0.7, 15 (pH6) 0.4, 8 (pH9) [43] XR 560 [44] -685 [44] 125…”

Automated QM/MM Screening of Rhodopsin Variants with Enhanced Fluorescence

“…1.1 × 10 –4 , extensive efforts have been made to search variants with enhanced fluorescence ( i.e. , via directed evolutionary approaches and random mutagenesis). − ,, Among others, variants such as QuasArs, Archons, , Archers, , Arch5, and Arch7 (see Table S1 in the Supporting Information) have been reported to feature a brighter fluorescence, enabling applications not only in the imaging of thin brain slices but also in living mammals and invertebrates. − Nevertheless, as shown in Table S2 in the Supporting Information, their ϕ f value remains in the range of 10 –3 –10 –2 and, therefore, not yet as bright as desirable.…”