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

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.

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

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.

Photocycle Dynamics of the Archaerhodopsin 3 Based Fluorescent Voltage Sensor QuasAr1.

The retinal photocycle dynamics of the fluorescent voltage sensor QuasAr1 (Archaerhodopsin 3 P60S-T80S-D95H-D106H-F161V mutant from Halorubrum sodomense) in pH 8 Tris buffer was studied. The samples were photoexcited to the first absorption band of the protonated retinal Schiff base (PRSB) Ret_580 (absorption maximum at λmax ≈ 580 nm), and the retinal Schiff base photoisomerization and protonation state changes were followed by absorption spectra recordings during light exposure and after light exposure. Ret_580 turned out to be composed of two protonated retinal Schiff base isomers, namely Ret_580I and Ret_580II. Photoexcitation of Ret_580I resulted in barrier-involved isomerization to Ret_540 (quantum yield ≈ 0.056) and subsequent retinal proton release leading to Ret_410 deprotonated retinal Schiff base (RSB). In the dark, Ret_410 partially recovered to Ret_580I and partially stabilized to irreversible Ret_400 due to apoprotein restructuring (Ret_410 lifetime ≈ 2 h). Photoexcitation of Ret_580II resulted in barrier-involved isomerization to Ret_640 (quantum yield ≈ 0.00135) and subsequent deprotonation to Ret_370 (RSB). In the dark, Ret_370 partially recovered to Ret_580II and partially stabilized to irreversible Ret_350 due to apoprotein restructuring (Ret_370 lifetime ≈ 10 h). Photocycle schemes and reaction coordinate diagrams for Ret_580I and Ret_580II were developed and photocyle parameters were determined.

Absorption and Emission Spectroscopic Investigation of Thermal Dynamics and Photo-Dynamics of the Rhodopsin Domain of the Rhodopsin-Guanylyl Cyclase from the Nematophagous Fungus Catenaria anguillulae.

The rhodopsin-guanylyl cyclase from the nematophagous fungus Catenaria anguillulae belongs to a recently discovered class of enzymerhodopsins and may find application as a tool in optogenetics. Here the rhodopsin domain CaRh of the rhodopsin-guanylyl cyclase from Catenaria anguillulae was studied by absorption and emission spectroscopic methods. The absorption cross-section spectrum and excitation wavelength dependent fluorescence quantum distributions of CaRh samples were determined (first absorption band in the green spectral region). The thermal stability of CaRh was studied by long-time attenuation measurements at room temperature (20.5 °C) and refrigerator temperature of 3.5 °C. The apparent melting temperature of CaRh was determined by stepwise sample heating up and cooling down (obtained apparent melting temperature: 62 ± 2 °C). The photocycle dynamics of CaRh was investigated by sample excitation to the first inhomogeneous absorption band of the CaRhda dark-adapted state around 590 nm (long-wavelength tail), 530 nm (central region) and 470 nm (short-wavelength tail) and following the absorption spectra development during exposure and after exposure (time resolution 0.0125 s). The original protonated retinal Schiff base PRSBall-trans in CaRhda photo-converted reversibly to protonated retinal Schiff base PRSBall-trans,la1 with restructured surroundings (CaRhla1 light-adapted state, slightly blue-shifted and broadened first absorption band, recovery to CaRhda with time constant of 0.8 s) and deprotonated retinal Schiff base RSB13-cis (CaRhla2 light-adapted state, first absorption band in violet to near ultraviolet spectral region, recovery to CaRhda with time constant of 0.35 s). Long-time light exposure of light-adapted CaRhla1 around 590, 530 and 470 nm caused low-efficient irreversible degradation to photoproducts CaRhprod. Schemes of the primary photocycle dynamics of CaRhda and the secondary photocycle dynamics of CaRhla1 are developed.

Key residues for the light regulation of the blue light-activated adenylyl cyclase from Beggiatoa sp.

Photoactivated adenylyl cyclases are powerful tools for optogenetics and for investigating signal transduction mechanisms in biological photoreceptors. Because of its large increase in enzyme activity in the light, the BLUF (blue light sensor using flavin adenine dinucleotide)-activated adenylyl cyclase (bPAC) from Beggiatoa sp. is a highly attractive model system for studying BLUF domain signaling. In this report, we studied the influence of site-directed mutations within the BLUF domain on the light regulation of the cyclase domain and determined key elements for signal transduction and color tuning. Photoactivation of the cyclase domain is accomplished via strand ß5 of the BLUF domain and involves the formation of helical structures in the cyclase domain as assigned by vibrational spectroscopy. In agreement with earlier studies, we observed severely impaired signaling in mutations directly on strand ß5 as well as in mutations affecting the hydrogen bond network around the flavin. Moreover, we identified a bPAC mutant with red-shifted absorbance and a decreased dark activity that is highly valuable for long-term optogenetic experiments. Additionally, we discovered a mutant that forms a stable neutral flavin semiquinone radical in the BLUF domain and surprisingly exhibits an inversion of light activation.

Reduction-oxidation photocycle dynamics of flavins in starch films.

The blue-light photo-reduction (conversion of oxidized flavin quinone via flavin semiquinone to fully reduced flavin hydroquinone) and dark re-oxidation of the flavins riboflavin and lumiflavin in starch (α-amylose) films was studied by absorption and luminescence spectroscopy. Blue-light photo-excitation caused an absorption, fluorescence, and phosphorescence decrease which recovered in the dark. The photo-reduction dark-oxidation cycle could be repeated. The efficiency of photo-reduction decreased with exposed excitation energy, and the speed of re-oxidation in the dark slowed down with time after excitation. The absorption did not fully recover. The fluorescence efficiency after a long time of storage in the dark increased beyond the initial flavin quinone fluorescence efficiency. Flavin photo-excitation is thought to cause starch-flavin restructuring (static fluorescence quenching center formation), enabling enhanced photo-induced starch to flavin electron transfer with subsequent flavin reduction and starch oxidation. In the dark, after light switch-off, thermal reversion of flavin reduction and starch oxidation occurred.

Photocycle dynamics of the Archaerhodopsin 3 based fluorescent voltage sensor Archon2.

The retinal photocycle dynamics of the fluorescent voltage sensor Archon2 in pH 8 Tris buffer was studied. Archon2 is a mutant of Archaerhodopsin 3 (Arch) from Halorubrum sodomense obtained by a robotic multidimensional directed evolution approach (Archon2 = Arch T56P-P60S-T80P-D95H-T99S-T116I-F161V-T183I-L197I-A225C). The samples were photo-excited to the first absorption band of the protonated retinal Schiff base (PRSB) Ret_586 (absorption maximum at λmax = 586 nm, excitation wavelengths λexc = 590 nm and 632.8 nm). The photocycle dynamics were studied by recording absorption spectra during light exposure and after light exposure. Ret_586 photoisomerized to Ret_535 (main component) and Ret_485 (minor component). Ret_535 backward photoisomerized to Ret_586 in light-adapted state (named Ret_586la) and partly deprotonated to neutral retinal Schiff base (RSB) Ret_372 in light adapted state (named Ret_372la, same isomer form as Ret_535). After excitation light switch-off Ret_372la recovered to Ret_372 in dark-adapted state (Ret_372da) which slowly re-protonated to Ret_535, and Ret_535 slowly isomerized back to Ret_586 in dark-adapted state (Ret_586da). Photocycle schemes and reaction coordinate diagrams are developed and photocycle parameters are determined.

Photo-dynamics of photoactivated adenylyl cyclase TpPAC from the spirochete bacterium Turneriella parva strain H(T).

The photoactivated adenylyl cyclase TpPAC from the spirochete bacterium Turneriella parva was synthesized and the purified recombinant protein was characterized by biochemical and optical spectroscopic methods. TpPAC consists of a BLUF domain (BLUF = Blue Light sensor Using Flavin) and an adenylyl cyclase homology domain (CHD). A light induced cAMP cyclase activity of ≈ 53.3 nmolmg(-1)min(-1) was measured while in the dark the cyclase activity was approximately a factor of 240 lower. The photo-cycling dynamics of the BLUF domain of TpPAC was studied by absorption spectra, fluorescence quantum distribution, and fluorescence lifetime measurements. The quantum efficiency of BLUF domain signaling state formation was found to be ϕs ≈ 0.59. A three-component exponential recovery of the signaling state to the receptor state was observed with the time constants τrec,1 = 4.8s, τrec,2 = 34.2s, and τrec,3 = 293s at 21.3 °C. The protein thermal stability was studied by stepwise sample heating and cooling. An apparent TpPAC melting temperature of ϑm ≈ 46 °C was determined. The photo-degradation of TpPAC in the signaling state was studied by prolonged intense light exposure at 455 nm. An irreversible flavin photo-degradation was observed with quantum yield ϕD ≈ 8.7 × 10(-6).

Bistable retinal schiff base photodynamics of histidine kinase rhodopsin HKR1 from Chlamydomonas reinhardtii.

The photodynamics of the recombinant rhodopsin fragment of the histidine kinase rhodopsin HKR1 from Chlamydomonas reinhardtii was studied by absorption and fluorescence spectroscopy. The retinal cofactor of HKR1 exists in two Schiff base forms RetA and RetB. RetA is the deprotonated 13-cis-retinal Schiff base (RSB) absorbing in the UVA spectral region. RetB is the protonated all-trans RSB absorbing in the blue spectral region. Blue light exposure converts RetB fully to RetA. UVA light exposure converts RetA to RetB and RetB to RetA giving a mixture determined by their absorption cross sections and their conversion efficiencies. The quantum efficiencies of conversion of RetA to RetB and RetB to RetA were determined to be 0.096 ± 0.005 and 0.405 ± 0.01 respectively. In the dark thermal equilibration between RetA and RetB with dominant RetA content occurred with a time constant of about 3 days at room temperature. The fluorescence emission behavior of RetA and RetB was studied, and fluorescence quantum yields of ϕ(F) (RetA) = 0.00117 and ϕ(F) (RetB) = 9.4 × 10(-5) were determined. Reaction coordinate schemes of the photodynamics are developed.

Absorption and emission spectroscopic characterization of photo-dynamics of photoactivated adenylyl cyclase mutant bPAC-Y7F of Beggiatoa sp.

The photoactivated cyclase bPAC of the microbial mats bacterium Beggiatoa sp. consists of a BLUF domain and an adenylyl cyclase domain. It has strong activity of photo-induced cyclic adenylyl monophosphate (cAMP) formation and is therefore an important optogenetic tool in neuroscience applications. The SUMO-bPAC-Y7F mutant where Tyr-7 is replaced by Phe-7 in the BLUF domain has lost the typical BLUF domain photo-cycle dynamics. Instead, the investigated SUMO-bPAC-Y7F mutant consisted of three protein conformations with different triplet based photo-dynamics: (i) reversible flavin quinone (Fl) cofactor reduction to flavin semiquinone (FlH), (ii) reversible violet/near ultraviolet absorbing flavin photoproduct (FlA) formation, and (iii) irreversible red absorbing flavin photoproduct (FlC) formation. Absorption and emission spectroscopic measurements on SUMO-bPAC-Y7F were carried out before, during and after light exposure. Flavin photo-dynamics schemes are developed for the SUMO-bPAC-Y7F fractions performing photo-induced FlH, FlA, and FlC formation. Quantitative parameters of the flavin cofactor excitation, relaxation and recovery dynamics in SUMO-bPAC-Y7F are determined.

Absorption and emission spectroscopic characterization of lumichrome in aqueous solutions.

The spectroscopic behavior of lumichrome (7,8-dimethyl-alloxazine, LC) in aqueous solutions in a pH range from -1.08 to 14.6 is studied. Absorption spectra, fluorescence quantum distributions, quantum yields, and lifetimes are determined. The ionization stage of ground-state LC changes with rising pH from the cationic form (LCH(2)(+)) to the neutral form (LCH) with a mid-point pH of pK(c) ≈ -0.53, and to the anionic form (LC(-)) with a mid-point pH of pK(a) ≈ 12.5. Above pH 7 a partial ground-state tautomerization of LCH to 7,8-dimethyl-isoalloxazine (IAH) occurs by N1-N10 intra-molecular proton transfer. For pH > pK(a) ≈ 12.5 LCH and IAH change to the anionic forms LC(-) and IA(-), and above pH 14 LC(-) tautomerizes completely to IA(-). In the excited state some neutral lumichrome (LCH*) converts to cationic lumichrome (LCH(2)(+)) at low pH by proton transfer from H(3)O(+) to LCH*. No photoinduced excited-state tautomerization of lumichrome was observed. LCH for pH > 3 and IAH are reasonably fluorescent. The fluorescence efficiencies of LC(-) and IA(-) are lower than those of LCH and IAH. The fluorescence of LCH(2)(+) is strongly quenched likely by intra-molecular diabatic charge transfer and excited-state relaxation by potential surface touching with the ground state.