Nanosecond Transient IR Spectroscopy of Halorhodopsin in Living Cells.

The ability to track minute changes of a single amino acid residue in a cellular environment is causing a paradigm shift in the attempt to fully understand the responses of biomolecules that are highly sensitive to their environment. Detecting early protein dynamics in living cells is crucial to understanding their mechanisms, such as those of photosynthetic proteins. Here, we elucidate the light response of the microbial chloride pump NmHR from the marine bacterium Nonlabens marinus, located in the membrane of living Escherichia coli cells, using nanosecond time-resolved UV/vis and IR absorption spectroscopy over the time range from nanoseconds to seconds. Transient structural changes of the retinal cofactor and the surrounding apoprotein are recorded using light-induced time-resolved UV/vis and IR difference spectroscopy. Of particular note, we have resolved the kinetics of the transient deprotonation of a single cysteine residue during the photocycle of NmHR out of the manifold of molecular vibrations of the cells. These findings are of high general relevance, given the successful development of optogenetic tools from photoreceptors to interfere with enzymatic and neuronal pathways in living organisms using light pulses as a noninvasive trigger.

Membrane Protein Activity Induces Specific Molecular Changes in Nanodiscs Monitored by FTIR Difference Spectroscopy.

It is well known that lipids neighboring integral membrane proteins directly influence their function. The opposite effect is true as well, as membrane proteins undergo structural changes after activation and thus perturb the lipidic environment. Here, we studied the interaction between these molecular machines and the lipid bilayer by observing changes in the lipid vibrational bands via FTIR spectroscopy. Membrane proteins with different functionalities have been reconstituted into lipid nanodiscs: Microbial rhodopsins that act as light-activated ion pumps (the proton pumps NsXeR and UmRh1, and the chloride pump NmHR) or as sensors (NpSRII), as well as the electron-driven cytochrome c oxidase RsCcO. The effects of the structural changes on the surrounding lipid phase are compared to mechanically induced lateral tension exerted by the light-activatable lipid analogue AzoPC. With the help of isotopologues, we show that the ν(C = O) ester band of the glycerol backbone reports on changes in the lipids' collective state induced by mechanical changes in the transmembrane proteins. The perturbation of the nanodisc lipids seems to involve their phase and/or packing state. 13C-labeling of the scaffold protein shows that its structure also responds to the mechanical expansion of the lipid bilayer.

The World of Algae Reveals a Broad Variety of Cryptochrome Properties and Functions.

Algae are photosynthetic eukaryotic (micro-)organisms, lacking roots, leaves, and other organs that are typical for land plants. They live in freshwater, marine, or terrestrial habitats. Together with the cyanobacteria they contribute to about half of global carbon fixation. As primary producers, they are at the basis of many food webs and they are involved in biogeochemical processes. Algae are evolutionarily distinct and are derived either by primary (e.g., green and red algae) or secondary endosymbiosis (e.g., diatoms, dinoflagellates, and brown algae). Light is a key abiotic factor needed to maintain the fitness of algae as it delivers energy for photosynthesis, regulates algal cell- and life cycles, and entrains their biological clocks. However, excess light can also be harmful, especially in the ultraviolet range. Among the variety of receptors perceiving light information, the cryptochromes originally evolved as UV-A and blue-light receptors and have been found in all studied algal genomes so far. Yet, the classification, biophysical properties, wavelength range of absorbance, and biological functions of cryptochromes are remarkably diverse among algal species, especially when compared to cryptochromes from land plants or animals.

Real-Time Tracking of Proton Transfer from the Reactive Cysteine to the Flavin Chromophore of a Photosensing Light Oxygen Voltage Protein.

LOV (light oxygen voltage) proteins are photosensors ubiquitous to all domains of life. A variant of the short LOV protein from Dinoroseobacter shibae (DsLOV) exhibits an exceptionally fast photocycle. We performed time-resolved molecular spectroscopy on DsLOV-M49S and characterized the formation of the thio-adduct state with a covalent bond between the reactive cysteine (C72) and C4a of the FMN. By use of a tunable quantum cascade laser, the weak absorption change of the vibrational band of S-H stretching vibration of C57 was resolved with a time resolution of 10 ns. Deprotonation of C72 proceeded with a time constant of 12 µs which tallies the rise of the thio-adduct state. These results provide valuable information for the mechanistic interpretation of light-induced structural changes in LOV domains, which involves the choreographed sequence of proton transfers, changes in electron density distributions, spin alterations of the latter, and transient bond formation and breakage. Such molecular insight will help develop new optogenetic tools based on flavin photoreceptors.

DASH cryptochrome 1, a UV-A receptor, balances the photosynthetic machinery of Chlamydomonas reinhardtii.

Drosophila, Arabidopsis, Synechocystis, Homo (DASH) cryptochromes belong to the cryptochrome/photolyase family and can act as DNA repair enzymes. In bacteria and fungi, they also can play regulatory roles, but in plants their biological functions remain elusive. Here, we characterize CRY-DASH1 from the green alga Chlamydomonas reinhardtii. We perform biochemical and in vitro photochemical analysis. For functional characterization, a knock-out mutant of cry-dash1 is used. CRY-DASH1 protein is localized in the chloroplast and accumulates at midday. Although the photoautotrophic growth of the mutant is significantly reduced compared to the wild-type (WT), the mutant has increased levels of photosynthetic pigments and a higher maximum photochemical efficiency of photosystem II (PS II). Hyper-stacking of thylakoid membranes occurs together with an increase in proteins of the PS II reaction center, D1 and its antenna CP43, but not of their transcripts. CRY-DASH1 binds fully reduced flavin adenine dinucleotide and the antenna 5,10-methenyltetrahydrofolate, leading to an absorption peak in the UV-A range. Supplementation of white light with UV-A increases photoautotrophic growth of the WT but not of the cry-dash1 mutant. These results suggest a balancing function of CRY-DASH1 in the photosynthetic machinery and point to its role as a photoreceptor for the UV-A range separated from the absorption of photosynthetic pigments.

Peripheral Methionine Residues Impact Flavin Photoreduction and Protonation in an Engineered LOV Domain Light Sensor.

Proton-coupled electron transfer reactions play critical roles in many aspects of sensory phototransduction. In the case of flavoprotein light sensors, reductive quenching of flavin excited states initiates chemical and conformational changes that ultimately transmit light signals to downstream targets. These reactions generally require neighboring aromatic residues and proton-donating side chains for rapid and coordinated electron and proton transfer to flavin. Although photoreduction of flavoproteins can produce either the anionic (ASQ) or neutral semiquinone (NSQ), the factors that favor one over the other are not well understood. Here we employ a biologically active variant of the light-oxygen-voltage (LOV) domain protein VVD devoid of the adduct-forming Cys residue (VVD-III) to probe the mechanism of flavin photoreduction and protonation. A series of isosteric and conservative residue replacements studied by rate measurements, fluorescence quantum yields, FTIR difference spectroscopy, and molecular dynamics simulations indicate that tyrosine residues facilitate charge recombination reactions that limit sustained flavin reduction, whereas methionine residues facilitate radical propagation and quenching and also gate solvent access for flavin protonation. Replacement of a single surface Met residue with Leu favors formation of the ASQ over the NSQ and desensitizes photoreduction to oxidants. In contrast, increasing site hydrophilicity by Gln substitution promotes rapid NSQ formation and weakens the influence of the redox environment. Overall, the photoreactivity of VVD-III can be understood in terms of redundant electron donors, internal hole quenching, and coupled proton transfer reactions that all depend upon protein conformation, dynamics, and solvent penetration.

Interconnection of the Antenna Pigment 8-HDF and Flavin Facilitates Red-Light Reception in a Bifunctional Animal-like Cryptochrome.

Cryptochromes are ubiquitous flavin-binding light sensors closely related to DNA-repairing photolyases. The animal-like cryptochrome CraCRY from the green alga Chlamydomonas reinhardtii challenges the paradigm of cryptochromes as pure blue-light receptors by acting as a (6-4) photolyase, using 8-hydroxy-5-deazaflavin (8-HDF) as a light-harvesting antenna with a 17.4 Šdistance to flavin and showing spectral sensitivity up to 680 nm. The expanded action spectrum is attributed to the presence of the flavin neutral radical (FADH•) in the dark, despite a rapid FADH• decay observed in vitro in samples exclusively carrying flavin. Herein, the red-light response of CraCRY carrying flavin and 8-HDF was studied, revealing a 3-fold prolongation of the FADH• lifetime in the presence of 8-HDF. Millisecond time-resolved ultraviolet-visible spectroscopy showed the red-light-induced formation and decay of an absorbance band at 458 nm concomitant with flavin reduction. Time-resolved Fourier transform infrared (FTIR) spectroscopy and density functional theory attributed these changes to the deprotonation of 8-HDF, challenging the paradigm of 8-HDF being permanently deprotonated in photolyases. FTIR spectra showed changes in the hydrogen bonding network of asparagine 395, a residue suggested to indirectly control flavin protonation, indicating the involvement of N395 in the stabilization of FADH•. Fluorescence spectroscopy revealed a decrease in the energy transfer efficiency of 8-HDF upon flavin reduction, possibly linked to 8-HDF deprotonation. The discovery of the interdependence of flavin and 8-HDF beyond energy transfer processes highlights the essential role of the antenna, introducing a new concept enabling CraCRY and possibly other bifunctional cryptochromes to fulfill their dual function.

Time-Resolved Infrared and Visible Spectroscopy on Cryptochrome aCRY: Basis for Red Light Reception.

Cryptochromes function as flavin-binding photoreceptors in bacteria, fungi, algae, land plants, and insects. The discovery of an animal-like cryptochrome in the green alga Chlamydomonas reinhardtii has expanded the spectral range of sensitivity of these receptors from ultraviolet A/blue light to almost the complete visible spectrum. The broadened light response has been explained by the presence of the flavin neutral radical as a chromophore in the dark. Concomitant with photoconversion of the flavin, an unusually long-lived tyrosyl radical with a red-shifted ultraviolet-visible spectrum is formed, which is essential for the function of the receptor. In this study, the microenvironment of this key residue, tyrosine 373, was scrutinized using time-resolved Fourier transform infrared spectroscopy on several variants of animal-like cryptochrome and density functional theory for band assignment. The reduced tyrosine takes on distinct hydrogen bond scenarios depending on the presence of the C-terminal extension and of a neighboring cysteine. Upon radical formation, all variants showed a signal at 1400 cm-1, which we assigned to the ν7'a marker band of the CO stretching mode. The exceptionally strong downshift of this band cannot be attributed to a loss of hydrogen bonding only. Time-resolved ultraviolet-visible spectroscopy on W322F, a mutant of the neighboring tryptophan residue, revealed a decrease of the tyrosyl radical lifetime by almost two orders of magnitude, along with a shift of the absorbance maximum from 416 to 398 nm. These findings strongly support the concept of a π-π stacking as an apolar interaction between Y373 and W322 to be responsible for the characteristics of the tyrosyl radical. This concept of radical stabilization has been unknown to cryptochromes so far but might be highly relevant for other homologs with a tetrad of tryptophans and tyrosines as electron donors.

Time-Resolved Infrared Spectroscopy on Plant Cryptochrome-Relevance of Proton Transfer and ATP Binding for Signaling.

Plant cryptochromes are light receptors in land plants and algae with very diverse functions such as circadian timing and lifecycle progression. The receptor consists of a photolyase homology region (PHR) binding the flavin chromophore and a C-terminal extension (CCT) responsible for signaling. The reputed signaling state, the flavin neutral radical, is formed by a femtosecond electron transfer and microsecond proton transfer to the excited, oxidized flavin. Subsequently, a 500 µs loss of ß-sheet structure ∼25 Å away from flavin was resolved and suggested to be part of the signal conduction to the CCT. Here, we performed time-resolved, step-scan Fourier transform IR spectroscopy on the PHR of the plant cryptochrome pCRY (formerly CPH1) from Chlamydomonas reinhardtii. In a mutant lacking the proton donor aspartic acid 396 only the flavin anion radical is formed, but we observed the loss of ß-sheet structure with a time constant of 1.3 ms, similar to the 500 µs of the wild type. This finding implies that the anion radical may be considered signaling-competent. In the steady state, a variation of external pH up to 8.3 did not have any effect on the difference spectra including the protonated state of Asp396. However, we detected the prominent loss of ß-sheet structure by illumination only in the presence of adenosine triphosphate (ATP). We conclude that the bound ATP stabilizes these light-induced changes in secondary structure to ensure a physiological lifetime compatible with signaling by plant cryptochrome.

Cryptochrome photoreceptors in green algae: Unexpected versatility of mechanisms and functions.

Green algae have a highly complex and diverse set of cryptochrome photoreceptor candidates including members of the following subfamilies: plant, plant-like, animal-like, DASH and cryptochrome photolyase family 1 (CPF1). While some green algae encode most or all of them, others lack certain members. Here we present an overview about functional analyses of so far investigated cryptochrome photoreceptors from the green algae Chlamydomonas reinhardtii (plant and animal-like cryptochromes) and Ostreococcus tauri (CPF1) with regard to their biological significance and spectroscopic properties. Cryptochromes of both algae have been demonstrated recently to be involved to various extents in circadian clock regulation and in Chlamydomonas additionally in life cycle control. Moreover, CPF1 even performs light-driven DNA repair. The plant cryptochrome and CPF1 are UVA/blue light receptors, whereas the animal-like cryptochrome responds to almost the whole visible spectrum including red light. Accordingly, plant cryptochrome, animal-like cryptochrome and CPF1 differ fundamentally in their structural response to light as revealed by their visible and infrared spectroscopic signatures, and in the role of the flavin neutral radical acting as dark form or signaling state.

Essential Role of an Unusually Long-lived Tyrosyl Radical in the Response to Red Light of the Animal-like Cryptochrome aCRY.

Cryptochromes constitute a group of flavin-binding blue light receptors in bacteria, fungi, plants, and insects. Recently, the response of cryptochromes to light was extended to nearly the entire visible spectral region on the basis of the activity of the animal-like cryptochrome aCRY in the green alga Chlamydomonas reinhardtii This finding was explained by the absorption of red light by the flavin neutral radical as the dark state of the receptor, which then forms the anionic fully reduced state. In this study, time-resolved UV-visible spectroscopy on the full-length aCRY revealed an unusually long-lived tyrosyl radical with a lifetime of 2.6 s, which is present already 1 µs after red light illumination of the flavin radical. Mutational studies disclosed the tyrosine 373 close to the surface to form the long-lived radical and to be essential for photoreduction. This residue is conserved exclusively in the sequences of other putative aCRY proteins distinguishing them from conventional (6-4) photolyases. Size exclusion chromatography showed the full-length aCRY to be a dimer in the dark at 0.5 mm injected concentration with the C-terminal extension as the dimerization site. Upon illumination, partial oligomerization was observed via disulfide bridge formation at cysteine 482 in close proximity to tyrosine 373. The lack of any light response in the C-terminal extension as evidenced by FTIR spectroscopy differentiates aCRY from plant and Drosophila cryptochromes. These findings imply that aCRY might have evolved a different signaling mechanism via a light-triggered redox cascade culminating in photooxidation of a yet unknown substrate or binding partner.

Microsecond Deprotonation of Aspartic Acid and Response of the α/ß Subdomain Precede C-Terminal Signaling in the Blue Light Sensor Plant Cryptochrome.

Plant cryptochromes are photosensory receptors that regulate various central aspects of plant growth and development. These receptors consist of a photolyase homology region (PHR) carrying the oxidized flavin adenine dinucleotide (FAD) cofactor, and a cryptochrome C-terminal extension (CCT), which is essential for signaling. Absorption of blue/UVA light leads to formation of the FAD neutral radical as the likely signaling state, and ultimately activates the CCT. Little is known about the signal transfer from the flavin to the CCT. Here, we investigated the photoreaction of the PHR by time-resolved step-scan FT-IR spectroscopy complemented by UV-vis spectroscopy. The first spectrum at 500 ns shows major contributions from the FAD anion radical, which is demonstrated to then be protonated by aspartic acid 396 to the neutral radical within 3.5 µs. The analysis revealed the existence of three intermediates characterized by changes in secondary structure. A marked loss of ß-sheet structure is observed in the second intermediate evolving with a time constant of 500 µs. This change is accompanied by a conversion of a tyrosine residue, which is identified as the formation of a tyrosine radical in the UV-vis. The only ß-sheet in the PHR is located within the α/ß subdomain, ∼25 Å away from the flavin. This subdomain has been previously attributed a role as a putative antenna binding site, but is now suggested to have evolved to a component in the signaling of plant cryptochromes by mediating the interaction with the CCT.

Proton transfer to flavin stabilizes the signaling state of the blue light receptor plant cryptochrome.

Plant cryptochromes regulate the circadian rhythm, flowering time, and photomorphogenesis in higher plants as responses to blue light. In the dark, these photoreceptors bind oxidized FAD in the photolyase homology region (PHR). Upon blue light absorption, FAD is converted to the neutral radical state, the likely signaling state, by electron transfer via a conserved tryptophan triad and proton transfer from a nearby aspartic acid. Here we demonstrate, by infrared and time-resolved UV-visible spectroscopy on the PHR domain, that replacement of the aspartic acid Asp-396 with cysteine prevents proton transfer. The lifetime of the radical is decreased by 6 orders of magnitude. This short lifetime does not permit to drive conformational changes in the C-terminal extension that have been associated with signal transduction. Only in the presence of ATP do both the wild type and mutant form a long-lived radical state. However, in the mutant, an anion radical is formed instead of the neutral radical, as found previously in animal type I cryptochromes. Infrared spectroscopic experiments demonstrate that the light-induced conformational changes of the PHR domain are conserved in the mutant despite the lack of proton transfer. These changes are not detected in the photoreduction of the non-photosensory d-amino acid oxidase to the anion radical. In conclusion, formation of the anion radical is sufficient to generate a protein response in plant cryptochromes. Moreover, the intrinsic proton transfer is required for stabilization of the signaling state in the absence of ATP.