The Carbonyl Group in ß2 of the Carotenoid Tunes the Photocycle Kinetics in Orange Carotenoid Protein.

Adaptation to rapid environmental changes is crucial for maintaining optimal photosynthetic efficiency and is ultimately key to the survival of all photosynthetic organisms. Like most of them, cyanobacteria protect their photosynthetic apparatus against rapidly increasing light intensities by nonphotochemical quenching (NPQ). In cyanobacteria, NPQ is controlled by Orange Carotenoid Protein (OCP) photocycle. OCP is the only known photoreceptor that uses carotenoid for its light activation. How carotenoid drives and controls this unique photoactivation process is still unknown. However, understanding and potentially controlling the OCP photocycle may open up new possibilities for improving photosynthetic biomass. Here we investigate the effect of the carbonyl group in the ß2 ring of the carotenoid on the OCP photocycle. We report microsecond to minute OCP light activation kinetics and Arrhenius plots of the two OCP forms: Canthaxanthin-bound OCP (OCPCAN) and echinenone-bound OCP (OCPECH). The difference between the two carotenoids is the presence of a carbonyl group in the ß2-ring located in the N-terminal domain of the protein. A combination of temperature-dependent spectroscopy, flash photolysis, and pump-probe transient absorption allows us to report the previously unresolved OCP intermediate associated primarily with the absorption bleach (OCPB). OCPB dominates the photokinetics in the µs to subms time range for OCPCAN and in the µs to ms range for OCPECH. We show that in OCPCAN the OCP photocycle steps are always faster than in OCPECH: from 2 to almost 20 times depending on the step. These results suggest that the presence of the carbonyl group in the ß2-ring of the carotenoid accelerates the OCP photocycle.

Light-induced Trpin/Metout Switching During BLUF Domain Activation in ATP-bound Photoactivatable Adenylate Cyclase OaPAC.

The understanding of signal transduction mechanisms in photoreceptor proteins is essential for elucidating how living organisms respond to light as environmental stimuli. In this study, we investigated the ATP binding, photoactivation and signal transduction process in the photoactivatable adenylate cyclase from Oscillatoria acuminata (OaPAC) upon blue light excitation. Structural models with ATP bound in the active site of native OaPAC at cryogenic as well as room temperature are presented. ATP is found in one conformation at cryogenic- and in two conformations at ambient-temperature, and is bound in an energetically unfavorable conformation for the conversion to cAMP. However, FTIR spectroscopic experiments confirm that this conformation is the native binding mode in dark state OaPAC and that transition to a productive conformation for ATP turnover only occurs after light activation. A combination of time-resolved crystallography experiments at synchrotron and X-ray Free Electron Lasers sheds light on the early events around the Flavin Adenine Dinucleotide (FAD) chromophore in the light-sensitive BLUF domain of OaPAC. Early changes involve the highly conserved amino acids Tyr6, Gln48 and Met92. Crucially, the Gln48 side chain performs a 180° rotation during activation, leading to the stabilization of the FAD chromophore. Cryo-trapping experiments allowed us to investigate a late light-activated state of the reaction and revealed significant conformational changes in the BLUF domain around the FAD chromophore. In particular, a Trpin/Metout transition upon illumination is observed for the first time in the BLUF domain and its role in signal transmission via α-helix 3 and 4 in the linker region between sensor and effector domain is discussed.

Single Amino Acid Mutation Decouples Photochemistry of the BLUF Domain from the Enzymatic Function of OaPAC and Drives the Enzyme to a Switched-on State.

Photoactivated adenylate cyclases (PACs) are light-activated enzymes that combine a BLUF (blue-light using flavin) domain and an adenylate cyclase domain that are able to increase the levels of the important second messenger cAMP (cyclic adenosine monophosphate) upon blue-light excitation. The light-induced changes in the BLUF domain are transduced to the adenylate cyclase domain via a mechanism that has not yet been established. One critical residue in the photoactivation mechanism of BLUF domains, present in the vicinity of the flavin is the glutamine amino acid close to the N5 of the flavin. The role of this residue has been investigated extensively both experimentally and theoretically. However, its role in the activity of the photoactivated adenylate cyclase, OaPAC has never been addressed. In this work, we applied ultrafast transient visible and infrared spectroscopies to study the photochemistry of the Q48E OaPAC mutant. This mutation altered the primary electron transfer process and switched the enzyme into a permanent 'on' state, able to increase the cAMP levels under dark conditions compared to the cAMP levels of the dark-adapted state of the wild-type OaPAC. Differential scanning calorimetry measurements point to a less compact structure for the Q48E OaPAC mutant. The ensemble of these findings provide insight into the important elements in PACs and how their fine tuning may help in the design of optogenetic devices.

Time-resolved study on signaling pathway of photoactivated adenylate cyclase and its nonlinear optical response.

Photoactivated adenylate cyclases (PACs) are multidomain BLUF proteins that regulate the cellular levels of cAMP in a light-dependent manner. The signaling route and dynamics of PAC from Oscillatoria acuminata (OaPAC), which consists of a light sensor BLUF domain, an adenylate cyclase domain, and a connector helix (α3-helix), were studied by detecting conformational changes in the protein moiety. Although circular dichroism and small-angle X-ray scattering measurements did not show significant changes upon light illumination, the transient grating method successfully detected light-induced changes in the diffusion coefficient (diffusion-sensitive conformational change (DSCC)) of full-length OaPAC and the BLUF domain with the α3-helix. DSCC of full-length OaPAC was observed only when both protomers in a dimer were photoconverted. This light intensity dependence suggests that OaPAC is a cyclase with a nonlinear light intensity response. The enzymatic activity indeed nonlinearly depends on light intensity, that is, OaPAC is activated under strong light conditions. It was also found that both DSCC and enzymatic activity were suppressed by a mutation in the W90 residue, indicating the importance of the highly conserved Trp in many BLUF domains for the function. Based on these findings, a reaction scheme was proposed together with the reaction dynamics.

Avoidance of the use of tryptophan in buried chromosomal proteins as a mechanism for reducing photo/oxidative damage to genomes.

In cells that are exposed to terrestrial sunlight, the indole moiety in the side chain of tryptophan (Trp) can suffer photo/oxidative damage (POD) by reactive oxygen species (ROS) and/or ultraviolet light (UV-B). Trp is oxidized to produce N-formylkynurenine (NFK), a UV-A-responsive photosensitizer that further degenerates into photosensitizers capable of generating ROS through exposure to visible light. Thus, Trp-containing proteins function as both victims, and perpetrators, of POD if they are not rapidly replaced through protein turnover. The literature indicates that protein turnover and DNA repair occur poorly in chromosomal interiors. We contend, therefore, that basic chromosomal proteins (BCPs) that are enveloped by DNA should have evolved to lack Trp residues in their amino acid sequences, since these could otherwise function as 'Trojan horse-type' DNA-damaging agents. Our global analyses of protein sequences demonstrates that BCPs consistently lack Trp residues, although DNA-binding proteins in general do not display such a lack. We employ HU-B (a wild-type, Trp-lacking bacterial BCP) and HU-B F47W (a mutant, Trp-containing form of the same bacterial BCP) to demonstrate that the possession of Trp is deleterious to BCPs and associated chromosomal DNA. Basically, we show that UV-B and UV-A (a) cause no POD in HU-B, but cause extensive POD in HU-B F47W (in vitro), as well as (b) only nominal DNA damage in bacteria expressing HU-B, but extensive DNA damage in bacteria expressing F47W HU-B (in vivo). Our results suggest that Trp-lacking BCPs could have evolved to reduce scope for protein-facilitated, sunlight-mediated damage of DNA by UV-A and visible light, within chromosomal interiors that are poorly serviced by protein turnover and DNA repair machinery.

Real-time detection of response regulator phosphorylation dynamics in live bacteria.

Bacteria utilize two-component system (TCS) signal transduction pathways to sense and adapt to changing environments. In a typical TCS, a stimulus induces a sensor histidine kinase (SHK) to phosphorylate a response regulator (RR), which then dimerizes and activates a transcriptional response. Here, we demonstrate that oligomerization-dependent depolarization of excitation light by fused mNeonGreen fluorescent protein probes enables real-time monitoring of RR dimerization dynamics in live bacteria. Using inducible promoters to independently express SHKs and RRs, we detect RR dimerization within seconds of stimulus addition in several model pathways. We go on to combine experiments with mathematical modeling to reveal that TCS phosphosignaling accelerates with SHK expression but decelerates with RR expression and SHK phosphatase activity. We further observe pulsatile activation of the SHK NarX in response to addition and depletion of the extracellular electron acceptor nitrate when the corresponding TCS is expressed from both inducible systems and the native chromosomal operon. Finally, we combine our method with polarized light microscopy to enable single-cell measurements of RR dimerization under changing stimulus conditions. Direct in vivo characterization of RR oligomerization dynamics should enable insights into the regulation of bacterial physiology.

Benchmarking of Cph1 Mutants and DrBphP for Light-Responsive Phytochrome-Based Hydrogels with Reversibly Adjustable Mechanical Properties.

In the rapidly expanding field of molecular optogenetics, the performance of the engineered systems relies on the switching properties of the underlying genetically encoded photoreceptors. In this study, the bacterial phytochromes Cph1 and DrBphP are engineered, recombinantly produced in Escherichia coli, and characterized regarding their switching properties in order to synthesize biohybrid hydrogels with increased light-responsive stiffness modulations. The R472A mutant of the cyanobacterial phytochrome 1 (Cph1) is identified to confer the phytochrome-based hydrogels with an increased dynamic range for the storage modulus but a different light-response for the loss modulus compared to the original Cph1-based hydrogel. Stiffness measurements of human atrial fibroblasts grown on these hydrogels suggest that differences in the loss modulus at comparable changes in the storage modulus affect cell stiffness and thus underline the importance of matrix viscoelasticity on cellular mechanotransduction. The hydrogels presented here are of interest for analyzing how mammalian cells respond to dynamic viscoelastic cues. Moreover, the Cph1-R472A mutant, as well as the benchmarking of the other phytochrome variants, are expected to foster the development and performance of future optogenetic systems.

A Systematic Survey of the Light/Dark-dependent Protein Degradation Events in a Model Cyanobacterium.

Light is essential for photosynthetic organisms and is involved in the regulation of protein synthesis and degradation. The significance of light-regulated protein degradation is exemplified by the well-established light-induced degradation and repair of the photosystem II reaction center D1 protein in higher plants and cyanobacteria. However, systematic studies of light-regulated protein degradation events in photosynthetic organisms are lacking. Thus, we conducted a large-scale survey of protein degradation under light or dark conditions in the model cyanobacterium Synechocystis sp. PCC 6803 (hereafter referred to as Synechocystis) using the isobaric labeling-based quantitative proteomics technique. The results revealed that 79 proteins showed light-regulated degradation, including proteins involved in photosystem II structure or function, quinone binding, and NADH dehydrogenase. Among these, 25 proteins were strongly dependent on light for degradation. Moreover, the light-dependent degradation of several proteins was sensitive to photosynthetic electron transport inhibitors (DCMU and DBMIB), suggesting that they are influenced by the redox state of the plastoquinone (PQ) pool. Together, our study comprehensively cataloged light-regulated protein degradation events, and the results serve as an important resource for future studies aimed at understanding light-regulated processes and protein quality control mechanisms in cyanobacteria.

The small acid-soluble proteins of Clostridioides difficile are important for UV resistance and serve as a check point for sporulation.

Clostridioides difficile is a nosocomial pathogen which causes severe diarrhea and colonic inflammation. C. difficile causes disease in susceptible patients when endospores germinate into the toxin-producing vegetative form. The action of these toxins results in diarrhea and the spread of spores into the hospital and healthcare environments. Thus, the destruction of spores is imperative to prevent disease transmission between patients. However, spores are resilient and survive extreme temperatures, chemical exposure, and UV treatment. This makes their elimination from the environment difficult and perpetuates their spread between patients. In the model spore-forming organism, Bacillus subtilis, the small acid-soluble proteins (SASPs) contribute to these resistances. The SASPs are a family of small proteins found in all endospore-forming organisms, C. difficile included. Although these proteins have high sequence similarity between organisms, the role(s) of the proteins differ. Here, we investigated the role of the main α/ß SASPs, SspA and SspB, and two annotated putative SASPs, CDR20291_1130 and CDR20291_3080, in protecting C. difficile spores from environmental insults. We found that SspA is necessary for conferring spore UV resistance, SspB minorly contributes, and the annotated putative SASPs do not contribute to UV resistance. In addition, the SASPs minorly contribute to the resistance of nitrous acid. Surprisingly, the combined deletion of sspA and sspB prevented spore formation. Overall, our data indicate that UV resistance of C. difficile spores is dependent on SspA and that SspA and SspB regulate/serve as a checkpoint for spore formation, a previously unreported function of SASPs.

Investigating the rus and petI operon expression patterns in exposed Acidithiobacillus ferrooxidans sp. FJ2 to different doses of gamma irradiation.

The bioleaching process is developing as an economic and successful biotechnology method in the metallurgy industry. Acidithiobacillus ferrooxidans is one of the most important bacteria involved in uranium bioleaching which converts insoluble U4+ to soluble U6+ by oxidation of Fe2+ to Fe3+ using several periplasmic proteins encoded by the genes in rus and petI operons in its electron transport pathway. Accordingly, the purpose of this study was to consider the expression of these genes through exposed A. ferrooxidans sp. FJ2 to γ-ray in 17 different doses targeting uranium extraction yield. Acidithiobacillus ferrooxidans sp. FJ2 was irradiated by gamma rays at 25, 50, 75, 100, 150, 300, 450, 600, 750 Gy and 1, 2, 5, 10, 15, 20, 25 and 30 kGy doses. Moreover, the Eh value of 9k culture media was measured as special screening criteria to select the four treatments. The selected bacteria were cultured in 9k media, containing 50% uranium ore powder in the bioleaching process. Then, the value of pH & Eh of culture media, Fe2+ and uranium concentrations in 4, 8 and 13 day's period of incubation were measured. In followings, the expression levels of cyc1, cyc2, rus, coxB, petA, petB, petC and cycA genes at the end of each period were investigated by real-time PCR. Overall, all samples demonstrated a decrease in pH value and Fe2+ concentration and an increase in Eh value and U concentration in time intervals. The gamma irradiation in given doses raised the expression levels of all genes encoded in rus and petI operons, except petB gene during the bioleaching process, although, it had no effect either on the pH, Eh values or on Fe2+ and uranium concentrations. This result implies that during the oxidation of ferrous iron and formation of Jarosite sediment, the decreasing trend of pH and the increasing trend of Eh occurred in all samples. However, the differences in expression of the genes of rus and petI operons in the samples did not have an effect on uranium extraction.

Local Electric Field Changes during the Photoconversion of the Bathy Phytochrome Agp2.

Phytochromes switch between a physiologically inactive and active state via a light-induced reaction cascade, which is initiated by isomerization of the tetrapyrrole chromophore and leads to the functionally relevant secondary structure transition of a protein segment (tongue). Although details of the underlying cause-effect relationships are not known, electrostatic fields are likely to play a crucial role in coupling chromophores and protein structural changes. Here, we studied local electric field changes during the photoconversion of the dark state Pfr to the photoactivated state Pr of the bathy phytochrome Agp2. Substituting Tyr165 and Phe192 in the chromophore pocket by para-cyanophenylalanine (pCNF), we monitored the respective nitrile stretching modes in the various states of photoconversion (vibrational Stark effect). Resonance Raman and IR spectroscopic analyses revealed that both pCNF-substituted variants undergo the same photoinduced structural changes as wild-type Agp2. Based on a structural model for the Pfr state of F192pCNF, a molecular mechanical-quantum mechanical approach was employed to calculate the electric field at the nitrile group and the respective stretching frequency, in excellent agreement with the experiment. These calculations serve as a reference for determining the electric field changes in the photoinduced states of F192pCNF. Unlike F192pCNF, the nitrile group in Y165pCNF is strongly hydrogen bonded such that the theoretical approach is not applicable. However, in both variants, the largest changes of the nitrile stretching modes occur in the last step of the photoconversion, supporting the view that the proton-coupled restructuring of the tongue is accompanied by a change of the electric field.

Green Light-Controlled Gene Switch for Mammalian and Plant Cells.

The quest to engineer increasingly complex synthetic gene networks in mammalian and plant cells requires an ever-growing portfolio of orthogonal gene expression systems. To control gene expression, light is of particular interest due to high spatial and temporal resolution, ease of dosage and simplicity of administration, enabling increasingly sophisticated man-machine interfaces. However, the majority of applied optogenetic switches are crowded in the UVB, blue and red/far-red light parts of the optical spectrum, limiting the number of simultaneously applicable stimuli. This problem is even more pertinent in plant cells, in which UV-A/B, blue, and red light-responsive photoreceptors are already expressed endogenously. To alleviate these challenges, we developed a green light responsive gene switch, based on the light-sensitive bacterial transcription factor CarH from Thermus thermophilus and its cognate DNA operator sequence CarO. The switch is characterized by high reversibility, high transgene expression levels, and low leakiness, leading to up to 350-fold induction ratios in mammalian cells. In this chapter, we describe the essential steps to build functional components of the green light-regulated gene switch, followed by detailed protocols to quantify transgene expression over time in mammalian cells. In addition, we expand this protocol with a description of how the optogenetic switch can be implemented in protoplasts of A. thaliana.

Studies of multifunctional DNA polymerase I from the extremely radiation resistant Deinococcus radiodurans: Recombinant expression, purification and characterization of the full-length protein and its large fragment.

Deinococcus radiodurans is a bacterium with extreme resistance to desiccation and radiation. Although the origins of this extreme resistance have not been fully elucidated, an efficient DNA repair machinery that includes the enzyme DNA polymerase I, is potentially crucial as part of a protection mechanism. Here we have cloned and performed small, medium, and large-scale expression of full-length D. radiodurans DNA polymerase I (DrPolI) as well as the large/Klenow fragment (DrKlenow). We then carried out functional characterization of 5' exonuclease, DNA strand displacement and polymerase activities of these proteins using gel-based and molecular beacon-based biochemical assays. With the same expression and purification strategy, we got higher yield in the production of DrKlenow than of the full-length protein, approximately 2.5 mg per liter of culture. Moreover, we detected a prominent 5' exonuclease activity of DrPolI in vitro. This activity and, DrKlenow strand displacement and DNA polymerase activities are preferentially stimulated at pH 8.0-8.5 and are reduced by addition of NaCl. Interestingly, both protein variants are more thermostable at pH 6.0-6.5. The characterization of DrPolI's multiple functions provides new insights into the enzyme's role in DNA repair pathways, and how the modulation of these functions is potentially used by D. radiodurans as a survival strategy.

Smart-watch-programmed green-light-operated percutaneous control of therapeutic transgenes.

Wearable smart electronic devices, such as smart watches, are generally equipped with green-light-emitting diodes, which are used for photoplethysmography to monitor a panoply of physical health parameters. Here, we present a traceless, green-light-operated, smart-watch-controlled mammalian gene switch (Glow Control), composed of an engineered membrane-tethered green-light-sensitive cobalamin-binding domain of Thermus thermophilus (TtCBD) CarH protein in combination with a synthetic cytosolic TtCBD-transactivator fusion protein, which manage translocation of TtCBD-transactivator into the nucleus to trigger expression of transgenes upon illumination. We show that Apple-Watch-programmed percutaneous remote control of implanted Glow-controlled engineered human cells can effectively treat experimental type-2 diabetes by producing and releasing human glucagon-like peptide-1 on demand. Directly interfacing wearable smart electronic devices with therapeutic gene expression will advance next-generation personalized therapies by linking biopharmaceutical interventions to the internet of things.

Scalable Three-Dimensional Photobioelectrodes Made of Reduced Graphene Oxide Combined with Photosystem I.

Photobioelectrodes represent one of the examples where artificial materials are combined with biological entities to undertake semi-artificial photosynthesis. Here, an approach is described that uses reduced graphene oxide (rGO) as an electrode material. This classical 2D material is used to construct a three-dimensional structure by a template-based approach combined with a simple spin-coating process during preparation. Inspired by this novel material and photosystem I (PSI), a biophotovoltaic electrode is being designed and investigated. Both direct electron transfer to PSI and mediated electron transfer via cytochrome c from horse heart as redox protein can be confirmed. Electrode preparation and protein immobilization have been optimized. The performance can be upscaled by adjusting the thickness of the 3D electrode using different numbers of spin-coating steps during preparation. Thus, photocurrents up to ∼14 µA/cm2 are measured for 12 spin-coated layers of rGO corresponding to a turnover frequency of 30 e- PSI-1 s-1 and external quantum efficiency (EQE) of 0.07% at a thickness of about 15 µm. Operational stability has been analyzed for several days. Particularly, the performance at low illumination intensities is very promising (1.39 µA/cm2 at 0.1 mW/cm2 and -0.15 V vs Ag/AgCl; EQE 6.8%).

Low-temperature Raman spectroscopy of sodium-pump rhodopsin from Indibacter alkaliphilus: insight of Na+ binding for active Na+ transport.

We carried out the low-temperature Raman measurement of a sodium pump rhodopsin from Indibacter alkaliphilus (IaNaR) and examined the primary structural change for the light-driven Na+ pump. We observed that photoexcitation of IaNaR produced the distorted 13-cis retinal chromophore in the presence of Na+, while the structural distortion was significantly relaxed in the absence of Na+. This structural difference of the chromophore with/without Na+ was attributed to the Na+ binding to the protein, which alters the active site. Using the spectral sensitivity to the ion binding, we found that IaNaR had a second Na+ binding site in addition to the one already specified on the extracellular surface. To date, the Na+ binding has not been considered as a prerequisite for Na+ transport. However, this study provides insight that the protein structural change induced by the ion binding involved the formation of an R108-D250 salt bridge, which has critical importance in the active transport of Na+.

Psb35 Protein Stabilizes the CP47 Assembly Module and Associated High-Light Inducible Proteins during the Biogenesis of Photosystem II in the Cyanobacterium Synechocystis sp. PCC6803.

Photosystem II (PSII) is a large membrane protein complex performing primary charge separation in oxygenic photosynthesis. The biogenesis of PSII is a complicated process that involves a coordinated linking of assembly modules in a precise order. Each such module consists of one large chlorophyll (Chl)-binding protein, number of small membrane polypeptides, pigments and other cofactors. We isolated the CP47 antenna module from the cyanobacterium Synechocystis sp. PCC 6803 and found that it contains a 11-kDa protein encoded by the ssl2148 gene. This protein was named Psb35 and its presence in the CP47 module was confirmed by the isolation of FLAG-tagged version of Psb35. Using this pulldown assay, we showed that the Psb35 remains attached to CP47 after the integration of CP47 into PSII complexes. However, the isolated Psb35-PSIIs were enriched with auxiliary PSII assembly factors like Psb27, Psb28-1, Psb28-2 and RubA while they lacked the lumenal proteins stabilizing the PSII oxygen-evolving complex. In addition, the Psb35 co-purified with a large unique complex of CP47 and photosystem I trimer. The absence of Psb35 led to a lower accumulation and decreased stability of the CP47 antenna module and associated high-light-inducible proteins but did not change the growth rate of the cyanobacterium under the variety of light regimes. Nevertheless, in comparison with WT, the Psb35-less mutant showed an accelerated pigment bleaching during prolonged dark incubation. The results suggest an involvement of Psb35 in the life cycle of cyanobacterial Chl-binding proteins, especially CP47.

Active-Site Controlled, Jahn-Teller Enabled Regioselectivity in Reductive S-C Bond Cleavage of S-Adenosylmethionine in Radical SAM Enzymes.

Catalysis by canonical radical S-adenosyl-l-methionine (SAM) enzymes involves electron transfer (ET) from [4Fe-4S]+ to SAM, generating an R3S0 radical that undergoes regioselective homolytic reductive cleavage of the S-C5' bond to generate the 5'-dAdo· radical. However, cryogenic photoinduced S-C bond cleavage has regioselectively yielded either 5'-dAdo· or ·CH3, and indeed, each of the three SAM S-C bonds can be regioselectively cleaved in an RS enzyme. This diversity highlights a longstanding central question: what controls regioselective homolytic S-C bond cleavage upon SAM reduction? We here provide an unexpected answer, founded on our observation that photoinduced S-C bond cleavage in multiple canonical RS enzymes reveals two enzyme classes: in one, photolysis forms 5'-dAdo·, and in another it forms ·CH3. The identity of the cleaved S-C bond correlates with SAM ribose conformation but not with positioning and orientation of the sulfonium center relative to the [4Fe-4S] cluster. We have recognized the reduced-SAM R3S0 radical is a (2E) state with its antibonding unpaired electron in an orbital doublet, which renders R3S0 Jahn-Teller (JT)-active and therefore subject to vibronically induced distortion. Active-site forces induce a JT distortion that localizes the odd electron in a single priority S-C antibond, which undergoes regioselective cleavage. In photolytic cleavage those forces act through control of the ribose conformation and are transmitted to the sulfur via the S-C5' bond, but during catalysis thermally induced conformational changes that enable ET from a cluster iron generate dominant additional forces that specifically select S-C5' for cleavage. This motion also can explain how 5'-dAdo· subsequently forms the organometallic intermediate Ω.

Photoreaction Dynamics of a Full-Length Protein YtvA and Intermolecular Interaction with RsbRA.

YtvA from Bacillus subtilis is a sensor protein that responds to blue light stress and regulates the activity of transcription factor σB. It is composed of the N-terminal LOV (light-oxygen-voltage) domain, the C-terminal STAS (sulfate transporter and anti-sigma factor antagonist) domain, and a linker region connecting them. In this study, the photoreaction and kinetics of full-length YtvA and the intermolecular interaction with a downstream protein, RsbRA, were revealed by the transient grating method. Although N-YLOV-linker, which is composed of the LOV domain of YtvA with helices A'α and Jα, exhibits a diffusion change due to the rotational motion of the helices, the YtvA dimer does not show the diffusion change. This result suggests that the STAS domain inhibits the rotational movement of helices A'α and Jα. We found that the YtvA dimer formed a heterotetramer with the RsbRA dimer probably via the interaction between the STAS domains, and we showed the diffusion change upon blue light illumination with a time constant faster than 70 µs. This result suggests a conformational change of the STAS domains; i.e., the interface between the STAS domains of the proteins changes to enhance the friction with water by the rotation structural change of helices A'α and Jα of YtvA.