Ultrafast terahertz Stark spectroscopy reveals the excited-state dipole moments of retinal in bacteriorhodopsin.

The photoinduced all-trans to 13-cis isomerization of the retinal Schiff base represents the ultrafast first step in the reaction cycle of bacteriorhodopsin (BR). Extensive experimental and theoretical work has addressed excited-state dynamics and isomerization via a conical intersection with the ground state. In conflicting molecular pictures, the excited state potential energy surface has been modeled as a pure S[Formula: see text] state that intersects with the ground state, or in a 3-state picture involving the S[Formula: see text] and S[Formula: see text] states. Here, the photoexcited system passes two crossing regions to return to the ground state. The electric dipole moment of the Schiff base in the S[Formula: see text] and S[Formula: see text] state differs strongly and, thus, its measurement allows for assessing the character of the excited-state potential. We apply the method of ultrafast terahertz (THz) Stark spectroscopy to measure electric dipole changes of wild-type BR and a BR D85T mutant upon electronic excitation. A fully reversible transient broadening and spectral shift of electronic absorption is induced by a picosecond THz field of several megavolts/cm and mapped by a 120-fs optical probe pulse. For both BR variants, we derive a moderate electric dipole change of 5 [Formula: see text] 1 Debye, which is markedly smaller than predicted for a neat S[Formula: see text]-character of the excited state. In contrast, S[Formula: see text]-admixture and temporal averaging of excited-state dynamics over the probe pulse duration gives a dipole change in line with experiment. Our results support a picture of electronic and nuclear dynamics governed by the interaction of S[Formula: see text] and S[Formula: see text] states in a 3-state model.

Multiple retinal isomerizations during the early phase of the bestrhodopsin photoreaction.

Bestrhodopsins constitute a class of light-regulated pentameric ion channels that consist of one or two rhodopsins in tandem fused with bestrophin ion channel domains. Here, we report on the isomerization dynamics in the rhodopsin tandem domains of Phaeocystis antarctica bestrhodopsin, which binds all-trans retinal Schiff-base (RSB) absorbing at 661 nm and, upon illumination, converts to the meta-stable P540 state with an unusual 11-cis RSB. The primary photoproduct P682 corresponds to a mixture of highly distorted 11-cis and 13-cis RSB directly formed from the excited state in 1.4 ps. P673 evolves from P682 in 500 ps and contains highly distorted 13-cis RSB, indicating that the 11-cis fraction in P682 converts to 13-cis. Next, P673 establishes an equilibrium with P595 in 1.2 µs, during which RSB converts to 11-cis and then further proceeds to P560 in 48 µs and P540 in 1.0 ms while remaining 11-cis. Hence, extensive isomeric switching occurs on the early ground state potential energy surface (PES) on the hundreds of ps to µs timescale before finally settling on a metastable 11-cis photoproduct. We propose that P682 and P673 are trapped high up on the ground-state PES after passing through either of two closely located conical intersections that result in 11-cis and 13-cis RSB. Co-rotation of C11=C12 and C13=C14 bonds results in a constricted conformational landscape that allows thermal switching between 11-cis and 13-cis species of highly strained RSB chromophores. Protein relaxation may release RSB strain, allowing it to evolve to a stable 11-cis isomeric configuration in microseconds.

The microbial opsin family of optogenetic tools.

The capture and utilization of light is an exquisitely evolved process. The single-component microbial opsins, although more limited than multicomponent cascades in processing, display unparalleled compactness and speed. Recent advances in understanding microbial opsins have been driven by molecular engineering for optogenetics and by comparative genomics. Here we provide a Primer on these light-activated ion channels and pumps, describe a group of opsins bridging prior categories, and explore the convergence of molecular engineering and genomic discovery for the utilization and understanding of these remarkable molecular machines.

Diversity of rhodopsin cyclases in zoospore-forming fungi.

Light perception for orientation in zoospore-forming fungi is linked to homo- or heterodimeric rhodopsin-guanylyl cyclases (RGCs). Heterodimeric RGCs, first identified in the chytrid Rhizoclosmatium globosum, consist of an unusual near-infrared absorbing highly fluorescent sensitizer neorhodopsin (NeoR) that is paired with a visual light-absorbing rhodopsin responsible for enzyme activation. Here, we present a comprehensive analysis of the distribution of RGC genes in early-branching fungi using currently available genetic data. Among the characterized RGCs, we identified red-sensitive homodimeric RGC variants with maximal light activation close to 600 nm, which allow for red-light control of GTP to cGMP conversion in mammalian cells. Heterodimeric RGC complexes have evolved due to a single gene duplication within the branching of Chytridiales and show a spectral range for maximal light activation between 480 to 600 nm. In contrast, the spectral sensitivity of NeoRs is reaching into the near-infrared range with maximal absorption between 641 and 721 nm, setting the low energy spectral edge of rhodopsins so far. Based on natural NeoR variants and mutational studies, we reevaluated the role of the counterion-triad proposed to cause the extreme redshift. With the help of chimera constructs, we disclose that the cyclase domain is crucial for functioning as homo- or heterodimers, which enables the adaptation of the spectral sensitivity by modular exchange of the photosensor. The extreme spectral plasticity of retinal chromophores in native photoreceptors provides broad perspectives on the achievable spectral adaptation for rhodopsin-based molecular tools ranging from UVB into the near-infrared.

A cytoplasmic protein kinase couples engagement of Chlamydomonas ciliary receptors to cAMP-dependent cellular responses.

The primary cilium is a cellular compartment specialized for receipt of extracellular signals that is essential for development and homeostasis. Although intraciliary responses to engagement of ciliary receptors are well studied, fundamental questions remain about the mechanisms and molecules that transduce ciliary signals into responses in the cytoplasm. During fertilization in the bi-ciliated alga Chlamydomonas reinhardtii, ciliary adhesion between plus and minus gametes triggers an immediate ∼10-fold increase in cellular cAMP and consequent responses in the cytoplasm required for cell-cell fusion. Here, we identify a new participant in ciliary signaling, Gamete-Specific Protein Kinase (GSPK). GSPK is essential for the adhesion-induced cAMP increase and for rapid gamete fusion. The protein is in the cytoplasm, and the entire cellular complement responds to a signal from the cilium by becoming phosphorylated within 1 min after ciliary receptor engagement. Unlike all other cytoplasmic events in ciliary signaling, GSPK phosphorylation is not responsive to exogenously added cAMP. Thus, during ciliary signaling in Chlamydomonas, a cytoplasmic protein is required to rapidly interpret a still uncharacterized ciliary signal to generate a cytoplasmic response.

Calredoxin regulates the chloroplast NADPH-dependent thioredoxin reductase in Chlamydomonas reinhardtii.

Calredoxin (CRX) is a calcium (Ca2+)-dependent thioredoxin (TRX) in the chloroplast of Chlamydomonas (Chlamydomonas reinhardtii) with a largely unclear physiological role. We elucidated the CRX functionality by performing in-depth quantitative proteomics of wild-type cells compared with a crx insertional mutant (IMcrx), two CRISPR/Cas9 KO mutants, and CRX rescues. These analyses revealed that the chloroplast NADPH-dependent TRX reductase (NTRC) is co-regulated with CRX. Electron transfer measurements revealed that CRX inhibits NADPH-dependent reduction of oxidized chloroplast 2-Cys peroxiredoxin (PRX1) via NTRC and that the function of the NADPH-NTRC complex is under strict control of CRX. Via non-reducing SDS-PAGE assays and mass spectrometry, our data also demonstrated that PRX1 is more oxidized under high light (HL) conditions in the absence of CRX. The redox tuning of PRX1 and control of the NADPH-NTRC complex via CRX interconnect redox control with active photosynthetic electron transport and metabolism, as well as Ca2+ signaling. In this way, an economic use of NADPH for PRX1 reduction is ensured. The finding that the absence of CRX under HL conditions severely inhibited light-driven CO2 fixation underpins the importance of CRX for redox tuning, as well as for efficient photosynthesis.

Insights into degradation and targeting of the photoreceptor channelrhodopsin-1.

In Chlamydomonas, the directly light-gated, plasma membrane-localized cation channels channelrhodopsins ChR1 and ChR2 are the primary photoreceptors for phototaxis. Their targeting and abundance is essential for optimal movement responses. However, our knowledge how Chlamydomonas achieves this is still at its infancy. Here we show that ChR1 internalization occurs via light-stimulated endocytosis. Prior or during endocytosis ChR1 is modified and forms high molecular mass complexes. These are the solely detectable ChR1 forms in extracellular vesicles and their abundance therein dynamically changes upon illumination. The ChR1-containing extracellular vesicles are secreted via the plasma membrane and/or the ciliary base. In line with this, ciliogenesis mutants exhibit increased ChR1 degradation rates. Further, we establish involvement of the cysteine protease CEP1, a member of the papain-type C1A subfamily. ΔCEP1-knockout strains lack light-induced ChR1 degradation, whereas ChR2 degradation was unaffected. Low light stimulates CEP1 expression, which is regulated via phototropin, a SPA1 E3 ubiquitin ligase and cyclic AMP. Further, mutant and inhibitor analyses revealed involvement of the small GTPase ARL11 and SUMOylation in ChR1 targeting to the eyespot and cilia. Our study thus defines the degradation pathway of this central photoreceptor of Chlamydomonas and identifies novel elements involved in its homoeostasis and targeting.

Cytokinin-deficient Chlamydomonas reinhardtii CRISPR-Cas9 mutants show reduced ability to prime resistance of tobacco against bacterial infection.

Although microalgae have only recently been recognized as part of the plant and soil microbiome, their application as biofertilizers has a tradition in sustainable crop production. Under consideration of their ability to produce the plant growth-stimulating hormone cytokinin (CK), known to also induce pathogen resistance, we have assessed the biocontrol ability of CK-producing microalgae. All pro- and eukaryotic CK-producing microalgae tested were able to enhance the tolerance of tobacco against Pseudomonas syringae pv. tabaci (PsT) infection. Since Chlamydomonas reinhardtii (Cre) proved to be the most efficient, we functionally characterized its biocontrol ability. We employed the CRISPR-Cas9 system to generate the first knockouts of CK biosynthetic genes in microalgae. Specifically, we targeted Cre Lonely Guy (LOG) and isopentenyltransferase (IPT) genes, the key genes of CK biosynthesis. While Cre wild-type exhibits a strong protection, the CK-deficient mutants have a reduced ability to induce plant defence. The degree of protection correlates with the CK levels, with the IPT mutants showing less protection than the LOG mutants. Gene expression analyses showed that Cre strongly stimulates tobacco resistance through defence gene priming. This study functionally verifies that Cre primes defence responses with CK, which contributes to the robustness of the effect. This work contributes to elucidate microalgae-mediated plant defence priming and identifies the role of CKs. In addition, these results underscore the potential of CK-producing microalgae as biologicals in agriculture by combining biofertilizer and biocontrol ability for sustainable and environment-friendly crop management.

Lysine acetylation regulates moonlighting activity of the E2 subunit of the chloroplast pyruvate dehydrogenase complex in Chlamydomonas.

The dihydrolipoamide acetyltransferase subunit DLA2 of the chloroplast pyruvate dehydrogenase complex (cpPDC) in the green alga Chlamydomonas reinhardtii has previously been shown to possess moonlighting activity in chloroplast gene expression. Under mixotrophic growth conditions, DLA2 forms part of a ribonucleoprotein particle (RNP) with the psbA mRNA that encodes the D1 protein of the photosystem II (PSII) reaction center. Here, we report on the characterization of the molecular switch that regulates shuttling of DLA2 between its functions in carbon metabolism and D1 synthesis. Determination of RNA-binding affinities by microscale thermophoresis demonstrated that the E3-binding domain (E3BD) of DLA2 mediates psbA-specific RNA recognition. Analyses of cpPDC formation and activity, as well as RNP complex formation, showed that acetylation of a single lysine residue (K197) in E3BD induces the release of DLA2 from the cpPDC, and its functional shift towards RNA binding. Moreover, Förster resonance energy transfer microscopy revealed that psbA mRNA/DLA2 complexes localize around the chloroplast's pyrenoid. Pulse labeling and D1 re-accumulation after induced PSII degradation strongly suggest that DLA2 is important for D1 synthesis during de novo PSII biogenesis.

Spectroscopic and Computational Observation of Glutamine Tautomerization in the Blue Light Sensing Using Flavin Domain Photoreaction.

Blue light sensing using flavin (BLUF) domains constitute a family of flavin-binding photoreceptors of bacteria and eukaryotic algae. BLUF photoactivation proceeds via a light-driven hydrogen-bond switch among flavin adenine dinucleotide (FAD) and glutamine and tyrosine side chains, whereby FAD undergoes electron and proton transfer with tyrosine and is subsequently re-oxidized by a hydrogen back-shuttle in picoseconds, constituting an important model system to understand proton-coupled electron transfer in biology. The specific structure of the hydrogen-bond patterns and the prevalence of glutamine tautomeric states in dark-adapted (DA) and light-activated (LA) states have remained controversial. Here, we present a combined femtosecond stimulated Raman spectroscopy (FSRS), computational chemistry, and site-selective isotope labeling Fourier-transform infrared spectroscopy (FTIR) study of the Slr1694 BLUF domain. FSRS showed distinct vibrational bands from the FADS1 singlet excited state. We observed small but significant shifts in the excited-state vibrational frequency patterns of the DA and LA states, indicating that these frequencies constitute a sensitive probe for the hydrogen-bond arrangement around FAD. Excited-state model calculations utilizing four different realizations of hydrogen bond patterns and glutamine tautomeric states were consistent with a BLUF reaction model that involved glutamine tautomerization to imidic acid, accompanied by a rotation of its side chain. A combined FTIR and double-isotope labeling study, with 13C labeling of FAD and 15N labeling of glutamine, identified the glutamine imidic acid C═N stretch vibration in the LA state and the Gln C═O in the DA state. Hence, our study provides support for glutamine tautomerization and side-chain rotation in the BLUF photoreaction.

Multifactorial in vivo regulation of the photoreceptor channelrhodopsin-1 abundance.

Oriented movement (phototaxis) is an efficient way to optimize light-driven processes and to avoid photodamage for motile algae. In Chlamydomonas the receptors for phototaxis are the channelrhodopsins ChR1 and ChR2. Both are directly light-gated, plasma membrane-localized cation channels. To optimally adjust its overall light-dependent responses, Chlamydomonas must tightly control the ChRs cellular abundance and integrate their activities into its general photoprotective network. How this is achieved is largely unknown. Here we show that the ChR1 protein level decreases upon illumination in a light-intensity and quality-dependent manner, whereas it is stable in prolonged darkness. Analysis of knockout strains of six major photoreceptors absorbing in the blue-violet range, which is most effective in evoking ChR1 degradation, revealed that only phototropin (PHOT) is involved. Notably, ChR2 degradation was normal in a ΔPHOT strain. Further, our results indicate that a COP1-SPA1 E3 ubiquitin ligase, the transcription factor Hy5 as well as changes in the cellular redox poise and cyclic nucleotide levels are additional components involved in this light acclimation response of Chlamydomonas. Our data highlight the presence of an adaptive framework connecting phototaxis with general photoprotective mechanisms via the use of overlapping signaling components already at the level of the primary photoreceptor.

Light-regulated adsorption and desorption of Chlamydomonas cells at surfaces.

Microbial colonization of surfaces represents the first step towards biofilm formation, which is a recurring phenomenon in nature with beneficial and detrimental implications in technological and medical settings. Consequently, there is interest in elucidating the fundamental aspects of the initial stages of biofilm formation of microorganisms on solid surfaces. While most of the research is oriented to understand bacterial surface colonization, the fundamental principles of surface colonization of motile, photosynthetic microbes remain largely unexplored so far. Recent single-cell studies showed that the flagellar adhesion of Chlamydomonas reinhardtii is switched on in blue light and switched off under red light [Kreis et al., Nat. Phys., 2018, 14, 45-49]. Here, we study this light-switchable surface association on the population level and measure the kinetics of adsorption and desorption of suspensions of motile C. reinhardtii cells on glass surfaces using bright-field optical microscopy. We observe that both processes exhibit a response lag relative to the time at which the blue- and red-light conditions are set and model this feature using time-delayed Langmuir-type kinetics. We find that cell adsorption occurs significantly faster than desorption, which we attribute to the protein-mediated molecular adhesion mechanism of the cells. Adsorption experiments using phototactically blind C. reinhardtii mutants demonstrate that phototaxis does not affect the cell adsorption kinetics. Hence, this framework can be used as an assay for characterizing the dynamics of the surface colonization of microbial species exhibiting light-regulated surface adhesion under precisely controlled environmental conditions.

Channelrhodopsin-1 Phosphorylation Changes with Phototactic Behavior and Responds to Physiological Stimuli in Chlamydomonas.

The unicellular alga Chlamydomonas (Chlamydomonas reinhardtii) exhibits oriented movement responses (phototaxis) to light over more than three log units of intensity. Phototaxis thus depends on the cell's ability to adjust the sensitivity of its photoreceptors to ambient light conditions. In Chlamydomonas, the photoreceptors for phototaxis are the channelrhodopsins (ChR)1 and ChR2; these light-gated cation channels are located in the plasma membrane. Although ChRs are widely used in optogenetic studies, little is known about ChR signaling in algae. We characterized the in vivo phosphorylation of ChR1. Its reversible phosphorylation occurred within seconds as a graded response to changes in the light intensity and ionic composition of the medium and depended on an elevated cytosolic Ca2+ concentration. Changes in the phototactic sign were accompanied by alterations in the phosphorylation status of ChR1. Furthermore, compared with the wild type, a permanently negative phototactic mutant required higher light intensities to evoke ChR1 phosphorylation. C-terminal truncation of ChR1 disturbed its reversible phosphorylation, whereas it was normal in ChR2-knockout and eyespot-assembly mutants. The identification of phosphosites in regions important for ChR1 function points to their potential regulatory role(s). We propose that multiple ChR1 phosphorylation, regulated via a Ca2+-based feedback loop, is an important component in the adaptation of phototactic sensitivity in Chlamydomonas.

A blue-light photoreceptor mediates the feedback regulation of photosynthesis.

In plants and algae, light serves both as the energy source for photosynthesis and a biological signal that triggers cellular responses via specific sensory photoreceptors. Red light is perceived by bilin-containing phytochromes and blue light by the flavin-containing cryptochromes and/or phototropins (PHOTs), the latter containing two photosensory light, oxygen, or voltage (LOV) domains. Photoperception spans several orders of light intensity, ranging from far below the threshold for photosynthesis to values beyond the capacity of photosynthetic CO2 assimilation. Excess light may cause oxidative damage and cell death, processes prevented by enhanced thermal dissipation via high-energy quenching (qE), a key photoprotective response. Here we show the existence of a molecular link between photoreception, photosynthesis, and photoprotection in the green alga Chlamydomonas reinhardtii. We show that PHOT controls qE by inducing the expression of the qE effector protein LHCSR3 (light-harvesting complex stress-related protein 3) in high light intensities. This control requires blue-light perception by LOV domains on PHOT, LHCSR3 induction through PHOT kinase, and light dissipation in photosystem II via LHCSR3. Mutants deficient in the PHOT gene display severely reduced fitness under excessive light conditions, indicating that the sensing, utilization, and dissipation of light is a concerted process that plays a vital role in microalgal acclimation to environments of variable light intensities.

Unifying photocycle model for light adaptation and temporal evolution of cation conductance in channelrhodopsin-2.

Although channelrhodopsin (ChR) is a widely applied light-activated ion channel, important properties such as light adaptation, photocurrent inactivation, and alteration of the ion selectivity during continuous illumination are not well understood from a molecular perspective. Herein, we address these open questions using single-turnover electrophysiology, time-resolved step-scan FTIR, and Raman spectroscopy of fully dark-adapted ChR2. This yields a unifying parallel photocycle model integrating now all so far controversial discussed data. In dark-adapted ChR2, the protonated retinal Schiff base chromophore (RSBH+) adopts an all-trans,C=N-anti conformation only. Upon light activation, a branching reaction into either a 13-cis,C=N-anti or a 13-cis,C=N-syn retinal conformation occurs. The anti-cycle features sequential H+ and Na+ conductance in a late M-like state and an N-like open-channel state. In contrast, the 13-cis,C=N-syn isomer represents a second closed-channel state identical to the long-lived P480 state, which has been previously assigned to a late intermediate in a single-photocycle model. Light excitation of P480 induces a parallel syn-photocycle with an open-channel state of small conductance and high proton selectivity. E90 becomes deprotonated in P480 and stays deprotonated in the C=N-syn cycle. Deprotonation of E90 and successive pore hydration are crucial for late proton conductance following light adaptation. Parallel anti- and syn-photocycles now explain inactivation and ion selectivity changes of ChR2 during continuous illumination, fostering the future rational design of optogenetic tools.

Modulation of Light Energy Transfer from Chromophore to Protein in the Channelrhodopsin ReaChR.

The function of photoreceptors relies on efficient transfer of absorbed light energy from the chromophore to the protein to drive conformational changes that ultimately generate an output signal. In retinal-binding proteins, mainly two mechanisms exist to store the photon energy after photoisomerization: 1) conformational distortion of the prosthetic group retinal, and 2) charge separation between the protonated retinal Schiff base (RSBH+) and its counterion complex. Accordingly, energy transfer to the protein is achieved by chromophore relaxation and/or reduction of the charge separation in the RSBH+-counterion complex. Combining FTIR and UV-Vis spectroscopy along with molecular dynamics simulations, we show here for the widely used, red-activatable Volvox carteri channelrhodopsin-1 derivate ReaChR that energy storage and transfer into the protein depends on the protonation state of glutamic acid E163 (Ci1), one of the counterions of the RSBH+. Ci1 retains a pKa of 7.6 so that both its protonated and deprotonated forms equilibrate at physiological conditions. Protonation of Ci1 leads to a rigid hydrogen-bonding network in the active-site region. This stabilizes the distorted conformation of the retinal after photoactivation and decelerates energy transfer into the protein by impairing the release of the strain energy. In contrast, with deprotonated Ci1 or removal of the Ci1 glutamate side chain, the hydrogen-bonded system is less rigid, and energy transfer by chromophore relaxation is accelerated. Based on the hydrogen out-of-plane (HOOP) band decay kinetics, we determined the activation energy for these processes in dependence of the Ci1 protonation state.

Dual Photoisomerization on Distinct Potential Energy Surfaces in a UV-Absorbing Rhodopsin.

UV-absorbing rhodopsins are essential for UV vision and sensing in all kingdoms of life. Unlike the well-known visible-absorbing rhodopsins, which bind a protonated retinal Schiff base for light absorption, UV-absorbing rhodopsins bind an unprotonated retinal Schiff base. Thus far, the photoreaction dynamics and mechanisms of UV-absorbing rhodopsins have remained essentially unknown. Here, we report the complete excited- and ground-state dynamics of the UV form of histidine kinase rhodopsin 1 (HKR1) from eukaryotic algae, using femtosecond stimulated Raman spectroscopy (FSRS) and transient absorption spectroscopy, covering time scales from femtoseconds to milliseconds. We found that energy-level ordering is inverted with respect to visible-absorbing rhodopsins, with an optically forbidden low-lying S1 excited state that has Ag- symmetry and a higher-lying UV-absorbing S2 state of Bu+ symmetry. UV-photoexcitation to the S2 state elicits a unique dual-isomerization reaction: first, C13═C14 cis-trans isomerization occurs during S2-S1 evolution in <100 fs. This very fast reaction features the remarkable property that the newly formed isomer appears in the excited state rather than in the ground state. Second, C15═N16 anti-syn isomerization occurs on the S1-S0 evolution to the ground state in 4.8 ps. We detected two ground-state unprotonated retinal photoproducts, 13-trans/15-anti (all-trans) and 13-cis/15-syn, after relaxation to the ground state. These isomers become protonated in 58 µs and 3.2 ms, respectively, resulting in formation of the blue-absorbing form of HKR1. Our results constitute a benchmark of UV-induced photochemistry of animal and microbial rhodopsins.

Targeting of Photoreceptor Genes in Chlamydomonas reinhardtii via Zinc-Finger Nucleases and CRISPR/Cas9.

The fast-growing biflagellated single-celled chlorophyte Chlamydomonas reinhardtii is the most widely used alga in basic research. The physiological functions of the 18 sensory photoreceptors are of particular interest with respect to Chlamydomonas development and behavior. Despite the demonstration of gene editing in Chlamydomonas in 1995, the isolation of mutants lacking easily ascertained newly acquired phenotypes remains problematic due to low DNA recombination efficiency. We optimized gene-editing protocols for several Chlamydomonas strains (including wild-type CC-125) using zinc-finger nucleases (ZFNs), genetically encoded CRISPR/associated protein 9 (Cas9) from Staphylococcus aureus and Streptococcus pyogenes, and recombinant Cas9 and developed protocols for rapidly isolating nonselectable gene mutants. Using this technique, we disrupted the photoreceptor genes COP1/2, COP3 (encoding channelrhodopsin 1 [ChR1]), COP4 (encoding ChR2), COP5, PHOT, UVR8, VGCC, MAT3, and aCRY and created the chr1 chr2 and uvr8 phot double mutants. Characterization of the chr1, chr2, and mat3 mutants confirmed the value of photoreceptor mutants for physiological studies. Genes of interest were disrupted in 5 to 15% of preselected clones (∼1 out of 4000 initial cells). Using ZFNs, genes were edited in a reliable, predictable manner via homologous recombination, whereas Cas9 primarily caused gene disruption via the insertion of cotransformed DNA. These methods should be widely applicable to research involving green algae.

Tip-Enhanced Infrared Difference-Nanospectroscopy of the Proton Pump Activity of Bacteriorhodopsin in Single Purple Membrane Patches.

Photosensitive proteins embedded in the cell membrane (about 5 nm thickness) act as photoactivated proton pumps, ion gates, enzymes, or more generally, as initiators of stimuli for the cell activity. They are composed of a protein backbone and a covalently bound cofactor (e.g. the retinal chromophore in bacteriorhodopsin (BR), channelrhodopsin, and other opsins). The light-induced conformational changes of both the cofactor and the protein are at the basis of the physiological functions of photosensitive proteins. Despite the dramatic development of microscopy techniques, investigating conformational changes of proteins at the membrane monolayer level is still a big challenge. Techniques based on atomic force microscopy (AFM) can detect electric currents through protein monolayers and even molecular binding forces in single-protein molecules but not the conformational changes. For the latter, Fourier-transform infrared spectroscopy (FTIR) using difference-spectroscopy mode is typically employed, but it is performed on macroscopic liquid suspensions or thick films containing large amounts of purified photosensitive proteins. In this work, we develop AFM-assisted, tip-enhanced infrared difference-nanospectroscopy to investigate light-induced conformational changes of the bacteriorhodopsin mutant D96N in single submicrometric native purple membrane patches. We obtain a significant improvement compared with the signal-to-noise ratio of standard IR nanospectroscopy techniques by exploiting the field enhancement in the plasmonic nanogap that forms between a gold-coated AFM probe tip and an ultraflat gold surface, as further supported by electromagnetic and thermal simulations. IR difference-spectra in the 1450-1800 cm-1 range are recorded from individual patches as thin as 10 nm, with a diameter of less than 500 nm, well beyond the diffraction limit for FTIR microspectroscopy. We find clear spectroscopic evidence of a branching of the photocycle for BR molecules in direct contact with the gold surfaces, with equal amounts of proteins either following the standard proton-pump photocycle or being trapped in an intermediate state not directly contributing to light-induced proton transport. Our results are particularly relevant for BR-based optoelectronic and energy-harvesting devices, where BR molecular monolayers are put in contact with metal surfaces, and, more generally, for AFM-based IR spectroscopy studies of conformational changes of proteins embedded in intrinsically heterogeneous native cell membranes.

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.