Color Tuning in Binding Pocket Models of the Chlamydomonas-Type Channelrhodopsins

We examined the shift of absorption maxima between the chlamydomonas-type channelrhodopsins (ChRs) and bacteriorhodopsin (BR). Starting from the BR X-ray structure, we modeled the color tuning in the binding pockets of the ChRs by mutating up to 28 amino acids in the vicinity of the chromophore. By applying the efficient self-consistent charge density functional tight binding (SCC-DFTB) method in a quantum mechanical/molecular mechanical (QM/MM) framework, including explicit polarization and calculating excitation energies with the semiempirical OM2/MRCI method and the ab initio SORCI method, we have shown that multiple mutations in the binding pocket of BR causes large hypsochromic shifts that are of the same order as the experimentally observed shifts of the absorption maxima between BR and the ChRs. This study further demonstrates that mutations in the proximity of the Schiff base and complex counterion lead to a stronger but more flexible interaction with the retinal, which could serve as a possible explanation for the spectral patterns found in the ChRs

Key Residues for the Light Regulation of the Blue Light-Activated Adenylyl Cyclase from <i>Beggiatoa</i> 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 <i>Beggiatoa</i> 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 β5 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 β5 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

Microbial and Animal Rhodopsins: Structures, Functions, and Molecular Mechanisms

Microbial and Animal Rhodopsins: Structures, Functions, and Molecular Mechanism

Unfolding of the C‑Terminal Jα Helix in the LOV2 Photoreceptor Domain Observed by Time-Resolved Vibrational Spectroscopy

Light-triggered reactions of biological photoreceptors have gained immense attention for their role as molecular switches in their native organisms and for optogenetic application. The light, oxygen, and voltage 2 (LOV2) sensing domain of plant phototropin binds a C-terminal Jα helix that is docked on a β-sheet and unfolds upon light absorption by the flavin mononucleotide (FMN) chromophore. In this work, the signal transduction pathway of LOV2 from Avena sativa was investigated using time-resolved infrared spectroscopy from picoseconds to microseconds. In D₂O buffer, FMN singlet-to-triplet conversion occurs in 2 ns and formation of the covalent cysteinyl-FMN adduct in 10 μs. We observe a two-step unfolding of the Jα helix: The first phase occurs concomitantly with Cys-FMN covalent adduct formation in 10 μs, along with hydrogen-bond rupture of the FMN C4O with Gln-513, motion of the β-sheet, and an additional helical element. The second phase occurs in approximately 240 μs. The final spectrum at 500 μs is essentially identical to the steady-state light-minus-dark Fourier transform infrared spectrum, indicating that Jα helix unfolding is complete on that time scale

Photoadduct Formation from the FMN Singlet Excited State in the LOV2 Domain of Chlamydomonas reinhardtii Phototropin

The two light, oxygen, and voltage domains of phototropin are blue-light photoreceptor domains that control various functions in plants and green algae. The key step of the light-driven reaction is the formation of a photoadduct between its FMN chromophore and a conserved cysteine, where the canonical reaction proceeds through the FMN triplet state. Here, complete photoreaction mapping of CrLOV2 from Chlamydomonas reinhardtii phototropin and AsLOV2 from Avena sativa phototropin-1 was realized by ultrafast broadband spectroscopy from femtoseconds to microseconds. We demonstrate that in CrLOV2, a direct photoadduct formation channel originates from the initially excited singlet state, in addition to the canonical reaction through the triplet state. This direct photoadduct reaction is coupled by a proton or hydrogen transfer process, as indicated by a significant kinetic isotope effect of 1.4 on the fluorescence lifetime. Kinetic model analyses showed that 38% of the photoadducts are generated from the singlet excited state

Hydrogen Bond Switching among Flavin and Amino Acids Determines the Nature of Proton-Coupled Electron Transfer in BLUF Photoreceptors

BLUF domains are flavin-binding photoreceptors that can be reversibly switched from a dark-adapted state to a light-adapted state. Proton-coupled electron transfer (PCET) from a conserved tyrosine to the flavin that results in a neutral flavin semiquinone/tyrosyl radical pair constitutes the photoactivation mechanism of BLUF domains. Whereas in the dark-adapted state PCET occurs in a sequential fashion where electron transfer precedes proton transfer, in the light-adapted state the same radical pair is formed by a concerted mechanism. We propose that the altered nature of the PCET process results from a hydrogen bond switch between the flavin and its surrounding amino acids that preconfigures the system for proton transfer. Hence, BLUF domains represent an attractive biological model system to investigate and understand PCET in great detail

Light–Dark Adaptation of Channelrhodopsin Involves Photoconversion between the all-<i>trans</i> and 13-<i>cis</i> Retinal Isomers

Channelrhodopsins (ChR) are light-gated ion channels of green algae that are widely used to probe the function of neuronal cells with light. Most ChRs show a substantial reduction in photocurrents during illumination, a process named “light adaptation”. The main objective of this spectroscopic study was to elucidate the molecular processes associated with light–dark adaptation. Here we show by liquid and solid-state nuclear magnetic resonance spectroscopy that the retinal chromophore of fully dark-adapted ChR is exclusively in an all<i>-trans</i> configuration. Resonance Raman (RR) spectroscopy, however, revealed that already low light intensities establish a photostationary equilibrium between all<i>-trans</i>,15<i>-anti</i> and 13<i>-cis</i>,15-<i>syn</i> configurations at a ratio of 3:1. The underlying photoreactions involve simultaneous isomerization of the C(13)C(14) and C(15)N bonds. Both isomers of this DA_app state may run through photoinduced reaction cycles initiated by photoisomerization of only the C(13)C(14) bond. RR spectroscopic experiments further demonstrated that photoinduced conversion of the apparent dark-adapted (DA_app) state to the photocycle intermediates P500 and P390 is distinctly more efficient for the all-<i>trans</i> isomer than for the 13-<i>cis</i> isomer, possibly because of different chromophore–water interactions. Our data demonstrating two complementary photocycles of the DA_app isomers are fully consistent with the existence of two conducting states that vary in quantitative relation during light–dark adaptation, as suggested previously by electrical measurements