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.

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.

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.

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.

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.

Evolution-inspired design of multicolored photoswitches from a single cyanobacteriochrome scaffold.

Cyanobacteriochromes (CBCRs) are small, bistable linear tetrapyrrole (bilin)-binding light sensors which are typically found as modular components in multidomain cyanobacterial signaling proteins. The CBCR family has been categorized into many lineages that roughly correlate with their spectral diversity, but CBCRs possessing a conserved DXCF motif are found in multiple lineages. DXCF CBCRs typically possess two conserved Cys residues: a first Cys that remains ligated to the bilin chromophore and a second Cys found in the DXCF motif. The second Cys often forms a second thioether linkage, providing a mechanism to sense blue and violet light. DXCF CBCRs have been described with blue/green, blue/orange, blue/teal, and green/teal photocycles, and the molecular basis for some of this spectral diversity has been well established. We here characterize AM1_1499g1, an atypical DXCF CBCR that lacks the second cysteine residue and exhibits an orange/green photocycle. Based on prior studies of CBCR spectral tuning, we have successfully engineered seven AM1_1499g1 variants that exhibit robust yellow/teal, green/teal, blue/teal, orange/yellow, yellow/green, green/green, and blue/green photocycles. The remarkable spectral diversity generated by modification of a single CBCR provides a good template for multiplexing synthetic photobiology systems within the same cellular context, thereby bypassing the time-consuming empirical optimization process needed for multiple probes with different protein scaffolds.

Structure of a Zinc Porphyrin-Substituted Bacterioferritin and Photophysical Properties of Iron Reduction.

The iron storage protein bacterioferritin (Bfr) binds up to 12 hemes b at specific sites in its protein shell. The heme b can be substituted with the photosensitizer Zn(II)-protoporphyrin IX (ZnPP), and photosensitized reductive iron release from the ferric oxyhydroxide {[FeO(OH)]n} core inside the ZnPP-Bfr protein shell was demonstrated [Cioloboc, D., et al. (2018) Biomacromolecules 19, 178-187]. This report describes the X-ray crystal structure of ZnPP-Bfr and the effects of loaded iron on the photophysical properties of the ZnPP. The crystal structure of ZnPP-Bfr shows a unique six-coordinate zinc in the ZnPP with two axial methionine sulfur ligands. Steady state and transient ultraviolet-visible absorption and luminescence spectroscopies show that irradiation with light overlapping the Soret absorption causes oxidation of ZnPP to the cation radical ZnPP•+ only when the ZnPP-Bfr is loaded with [FeO(OH)]n. Femtosecond transient absorption spectroscopy shows that this photooxidation occurs from the singlet excited state (1ZnPP*) on the picosecond time scale and is consistent with two oxidizing populations of Fe3+, which do not appear to involve the ferroxidase center iron. We propose that [FeO(OH)]n clusters at or near the inner surface of the protein shell are responsible for ZnPP photooxidation. Hopping of the photoinjected electrons through the [FeO(OH)]n would effectively cause migration of Fe2+ through the inner cavity to pores where it exits the protein. Reductive iron mobilization is presumed to be a physiological function of Bfrs. The phototriggered Fe3+ reduction could be used to identify the sites of iron mobilization within the Bfr protein shell.

Structural, Photophysical, and Photochemical Characterization of Zinc Protoporphyrin IX in a Dimeric Variant of an Iron Storage Protein: Insights into the Mechanism of Photosensitized H2 Generation.

Some of us have previously reported the preparation of a dimeric form of the iron storage protein, bacterioferritin (Bfr), in which the native heme b is substituted with the photosensitizer, Zn(II)-protoporphyrin IX (ZnPP-Bfr dimer). We further showed that the ZnPP-Bfr dimer can serve as a photosensitizer for platinum-catalyzed H2 generation in aqueous solution without the usually added electron relay between photosensitizer and platinum ( Clark , E. R. , Inorg. Chem. 2017 , 56 , 4584 - 4593 ). We proposed reductive or oxidative quenching pathways involving the ZnPP anion radical (ZnPP•-) or the ZnPP cation radical, (ZnPP•+), respectively. The present report describes structural, photophysical, and photochemical properties of the ZnPP in the ZnPP-Bfr dimer. X-ray absorption spectroscopic studies at 10 K showed a mixture of five- and six-coordinated Zn centers with axial coordination by one long Zn-SγMet distance of ∼2.8 Å and ∼40% having an additional shorter Zn-S distance of ∼2.4 Å, in addition to the expected 4 nitrogen atom coordination from the porphyrin. The ZnPP in ZnPP-Bfr dimer was prone to photosensitized oxidation to ZnPP•+. The ZnPP•+ was rapidly reduced by ascorbic acid, which we previously determined was essential for photosensitized H2 production in this system. These results are consistent with an oxidative quenching pathway involving electron transfer from 3ZnPP* to platinum, which may be assisted by a flexible ZnPP axial coordination sphere. However, the low quantum yield for H2 production (∼1%) in this system could make reductive quenching difficult to detect, and can, therefore, not be completely ruled out. The ZnPP-Bfr dimer provides a simple but versatile framework for mechanistic assessment and optimization of porphyrin-photosensitized H2 generation without an electron relay between porphyrin and the platinum catalyst.

A Dual Role for Ca2+ in Expanding the Spectral Diversity and Stability of Light-Harvesting 1 Reaction Center Photocomplexes of Purple Phototrophic Bacteria.

The light-harvesting 1 reaction center (LH1-RC) complex in the purple sulfur bacterium Thiorhodovibrio ( Trv.) strain 970 cells exhibits its LH1 Q y transition at 973 nm, the lowest-energy Q y absorption among purple bacteria containing bacteriochlorophyll a (BChl a). Here we characterize the origin of this extremely red-shifted Q y transition. Growth of Trv. strain 970 did not occur in cultures free of Ca2+, and elemental analysis of Ca2+-grown cells confirmed that purified Trv. strain 970 LH1-RC complexes contained Ca2+. The LH1 Q y band of Trv. strain 970 was blue-shifted from 959 to 875 nm upon Ca2+ depletion, but the original spectral properties were restored upon Ca2+ reconstitution, which also occurs with the thermophilic purple bacterium Thermochromatium ( Tch.) tepidum. The amino acid sequences of the LH1 α- and ß-polypeptides from Trv. strain 970 closely resemble those of Tch. tepidum; however, Ca2+ binding in the Trv. strain 970 LH1-RC occurred more selectively than in Tch. tepidum LH1-RC and with a reduced affinity. Ultraviolet resonance Raman analysis indicated that the number of hydrogen-bonding interactions between BChl a and LH1 proteins of Trv. strain 970 was significantly greater than for Tch. tepidum and that Ca2+ was indispensable for maintaining these bonds. Furthermore, perfusion-induced Fourier transform infrared analyses detected Ca2+-induced conformational changes in the binding site closely related to the unique spectral properties of Trv. strain 970. Collectively, our results reveal an ecological strategy employed by Trv. strain 970 of integrating Ca2+ into its LH1-RC complex to extend its light-harvesting capacity to regions of the near-infrared spectrum unused by other purple bacteria.

DdrO is an essential protein that regulates the radiation desiccation response and the apoptotic-like cell death in the radioresistant Deinococcus radiodurans bacterium.

Deinococcus radiodurans is known for its extreme radioresistance. Comparative genomics identified a radiation-desiccation response (RDR) regulon comprising genes that are highly induced after DNA damage and containing a conserved motif (RDRM) upstream of their coding region. We demonstrated that the RDRM sequence is involved in cis-regulation of the RDR gene ddrB in vivo. Using a transposon mutagenesis approach, we showed that, in addition to ddrO encoding a predicted RDR repressor and irrE encoding a positive regulator recently shown to cleave DdrO in Deinococcus deserti, two genes encoding α-keto-glutarate dehydrogenase subunits are involved in ddrB regulation. In wild-type cells, the DdrO cell concentration decreased transiently in an IrrE-dependent manner at early times after irradiation. Using a conditional gene inactivation system, we showed that DdrO depletion enhanced expression of three RDR proteins, consistent with the hypothesis that DdrO acts as a repressor of the RDR regulon. DdrO-depleted cells loose viability and showed morphological changes evocative of an apoptotic-like response, including membrane blebbing, defects in cell division and DNA fragmentation. We propose that DNA repair and apoptotic-like death might be two responses mediated by the same regulators, IrrE and DdrO, but differently activated depending on the persistence of IrrE-dependent DdrO cleavage.

Light-driven Na(+) pump from Gillisia limnaea: a high-affinity Na(+) binding site is formed transiently in the photocycle.

A group of microbial retinal proteins most closely related to the proton pump xanthorhodopsin has a novel sequence motif and a novel function. Instead of, or in addition to, proton transport, they perform light-driven sodium ion transport, as reported for one representative of this group (KR2) from Krokinobacter. In this paper, we examine a similar protein, GLR from Gillisia limnaea, expressed in Escherichia coli, which shares some properties with KR2 but transports only Na(+). The absorption spectrum of GLR is insensitive to Na(+) at concentrations of ≤3 M. However, very low concentrations of Na(+) cause profound differences in the decay and rise time of photocycle intermediates, consistent with a switch from a "Na(+)-independent" to a "Na(+)-dependent" photocycle (or photocycle branch) at ∼60 µM Na(+). The rates of photocycle steps in the latter, but not the former, are linearly dependent on Na(+) concentration. This suggests that a high-affinity Na(+) binding site is created transiently after photoexcitation, and entry of Na(+) from the bulk to this site redirects the course of events in the remainder of the cycle. A greater concentration of Na(+) is needed for switching the reaction path at lower pH. The data suggest therefore competition between H(+) and Na(+) to determine the two alternative pathways. The idea that a Na(+) binding site can be created at the Schiff base counterion is supported by the finding that upon perturbation of this region in the D251E mutant, Na(+) binds without photoexcitation. Binding of Na(+) to the mutant shifts the chromophore maximum to the red like that of H(+), which occurs in the photocycle of the wild type.

Light activation of Staphylococcus aureus toxin YoeBSa1 reveals guanosine-specific endoribonuclease activity.

The Staphylococcus aureus chromosome harbors two homologues of the YefM-YoeB toxin-antitoxin (TA) system. The toxins YoeBSa1 and YoeBSa2 possess ribosome-dependent ribonuclease (RNase) activity in Escherichia coli. This activity is similar to that of the E. coli toxin YoeBEc, an enzyme that, in addition to ribosome-dependent RNase activity, possesses ribosome-independent RNase activity in vitro. To investigate whether YoeBSa1 is also a ribosome-independent RNase, we expressed YoeBSa1 using a novel strategy and characterized its in vitro RNase activity, sequence specificity, and kinetics. Y88 of YoeBSa1 was critical for in vitro activity and cell culture toxicity. This residue was mutated to o-nitrobenzyl tyrosine (ONBY) via unnatural amino acid mutagenesis. YoeBSa1-Y88ONBY could be expressed in the absence of the antitoxin YefMSa1 in E. coli. Photocaged YoeBSa1-Y88ONBY displayed UV light-dependent RNase activity toward free mRNA in vitro. The in vitro ribosome-independent RNase activity of YoeBSa1-Y88ONBY, YoeBSa1-Y88F, and YoeBSa1-Y88TAG was significantly reduced or abolished. In contrast to YoeBEc, which cleaves RNA at both adenosine and guanosine with a preference for adenosine, YoeBSa1 cleaved mRNA specifically at guanosine. Using this information, a fluorometric assay was developed and used to determine the kinetic parameters for ribosome-independent RNA cleavage by YoeBSa1.

Screening of Lactobacillus strains for their ability to bind benzo(a)pyrene and the mechanism of the process.

In order to investigate the binding ability of Lactobacillus strains to Benzo(a)pyrene (BaP), 15 strains were analysed. L. plantarum CICC 22135 and L. pentosus CICC 23163 exhibited high efficiency in removing BaP from aqueous medium; the binding rates were 66.76% and 64.31%, respectively. This process was affected by temperature, incubation time and pH, and cell viability was not necessary for the binding ability. Additionally, both strains, especially strain CICC 23163 showed high specificity in binding BaP. The cell-BaP complexes were stable in aqueous medium. The mechanism of binding was investigated by examining the binding ability of different components of the microorganism cells. The results revealed that peptidoglycans played an important role in binding BaP and its structural integrity was required. Consequently, we proposed that the mechanism of this process was a physisorption and peptidoglycan was the main binding site. These two strains may be used for dietary detoxification in human diet and animal feed.

A circularly permuted photoactive yellow protein as a scaffold for photoswitch design.

Upon blue light irradiation, photoactive yellow protein (PYP) undergoes a conformational change that involves large movements at the N-terminus of the protein. We reasoned that this conformational change might be used to control other protein or peptide sequences if these were introduced as linkers connecting the N- and C-termini of PYP in a circular permutant. For such a design strategy to succeed, the circularly permuted PYP (cPYP) would have to fold normally and undergo a photocycle similar to that of the wild-type protein. We created a test cPYP by connecting the N- and C-termini of wild-type PYP (wtPYP) with a GGSGGSGG linker polypeptide and introducing new N- and C-termini at G115 and S114, respectively. Biophysical analysis indicated that this cPYP adopts a dark-state conformation much like wtPYP and undergoes wtPYP-like photoisomerization driven by blue light. However, thermal recovery of dark-state cPYP is ∼10-fold faster than that of wtPYP, so that very bright light is required to significantly populate the light state. Targeted mutations at M121E (M100 in wtPYP numbering) were found to enhance the light sensitivity substantially by lengthening the lifetime of the light state to ∼10 min. Nuclear magnetic resonance (NMR), circular dichroism, and UV-vis analysis indicated that the M121E-cPYP mutant also adopts a dark-state structure like that of wtPYP, although protonated and deprotonated forms of the chromophore coexist, giving rise to a shoulder near 380 nm in the UV-vis absorption spectrum. Fluorine NMR studies with fluorotryptophan-labeled M121E-cPYP show that blue light drives large changes in conformational dynamics and leads to solvent exposure of Trp7 (Trp119 in wtPYP numbering), consistent with substantial rearrangement of the N-terminal cap structure. M121E-cPYP thus provides a scaffold that may allow a wider range of photoswitchable protein designs via replacement of the linker polypeptide with a target protein or peptide sequence.

Effect of UV radiation on a thermostable superoxide dismutase purified from a thermophilic bacterium isolated from a sterilization drying oven.

A thermostable superoxide dismutase from a thermophilic bacterium, called Geobacillus wiegeli (GWE1), isolated from the interior of a sterilization drying oven, was purified by anion-exchange and molecular size-exclusion liquid chromatography. On the basis of SDS-PAGE, the purified enzyme was found to be homogeneous and showed an estimated subunit molecular mass of 23.9 kDa. The holoenzyme is a homotetramer of 97.3 kDa. Superoxide dismutase exhibited maximal activity at pH 8.5 and at temperature around 60 ºC. The enzyme was thermostable maintaining 50% of its activity even after 4.5 hours incubation at 60 ºC and more than 70% of its activity after 30 min at 80 ºC. When the microorganism was irradiated with UVA, an increase in the specific activity of superoxide dismutase was observed which was correlated with decreasing levels of anion superoxide, indicating the direct involvement of this enzyme in the capture of reactive oxygen species. This study reports the effects of UV radiation on a superoxide dismutase from a thermophilic bacterium isolated from an anthropogenic environment.

DR2417, a hypothetical protein characterized as a novel ß-CASP family nuclease in radiation resistant bacterium, Deinococcus radiodurans.

BACKGROUND: Deinococcus radiodurans survives extreme doses of radiations contributed by efficient DNA repair pathways. DR2417 (DncA) was detected separately both in a pool of nucleotide binding proteins and multiprotein complex isolated from cells undergoing DNA repair. SCOPE OF REVIEW: DR_2417m ORF was sequenced and amino acid sequence of DncA was search for structural similarities with other proteins and functional motifs. Recombinant DncA was characterized for its DNA metabolic functions in vitro and its role in radiation resistance. MAJOR CONCLUSIONS: Sequencing of DR_2417m did not show the reported frame shift at 996th nucleotide position of this gene. DncA showed similarities with ß-CASP family nucleases. Recombinant protein acted efficiently on dsDNA and showed an Mn2+ dependent 3'→5' exonuclease and ssDNA/dsDNA junction endonuclease activities while a very low level activity on RNA. The DNase activity of this protein was inhibited in presence of ATP. Its transcription was induced upon γ radiation exposure and a reduction in its copy number resulted in reduced growth rate and loss of γ radiation resistance in Deinococcus. CONCLUSION: Our results suggest that DncA was a novel nuclease of ß CASP family having a strong dsDNA end processing activity and it seems to be an essential gene required for both growth and γ radiation resistance of this bacterium. GENERAL SIGNIFICANCE: Traditionally DncA should have shown both DNase and RNase functions as other members of ß CASP family nucleases. A strong DNase and poor RNase activity possibly made it functionally significant in the radioresistance of D. radiodurans, which would be worth investigating independently.

Improvement of chaperone activity of 2-Cys peroxiredoxin using gamma ray.

A typical 2-cysteine peroxiredoxin (2-Cys Prx) PaPrx can act alternatively as thioredoxin (Trx)-dependent peroxidase and molecular chaperone in Pseudomonas aeruginosa PAO1. In addition, the functional switch of PaPrx is regulated by its structural change which is dependently induced by stress conditions. In the present study, we examined the effect of gamma ray on structural modification related to chaperone activity of PaPrx. The structural change of PaPrx occupied with gamma ray irradiation (2 kGy) based on polyacrylamide gel electrophoresis (PAGE) analysis and the functional change also began. The enhanced chaperone activity was increased about 3-4 folds at 30 kGy gamma irradiation compared with nonirradiated PaPrx, while the peroxidase activity was significantly decreased. We also investigated the influence of the gamma ray on protein hydrophobicity as related to chaperone function. The exposure of hydrophobic domains reached a peak at 30 kGy gamma ray and then decreased dependently with increasing gamma irradiation. Our results suggest that highly enhanced chaperone activity could be adapted for use in bio-engineering systems and industrial applications such as enzyme stabilization during industrial process (inactivation protection), improvement of useful protein productivity (refolding and secretion) and industrial animal cell cultivation (stress protection).

Coupling between the BLUF and EAL domains in the blue light-regulated phosphodiesterase BlrP1.

The first biochemical and structural characterization of the full-length active photoreceptor BlrP1 from Klebsiella pneumoniae was recently reported by Barends et al. [Nature 459:1015-1018, (2009)]. The light-regulated catalytic function of its C-terminal c-di-guanosine monophosphate phosphodiesterase, the EAL (Glu-Ala-Leu) domain, is activated by the N-terminal sensor of blue light using the flavin adenine dinucleotide (BLUF) domain. We performed molecular dynamics simulations on the dimeric BlrP1 protein in order to examine the coupling regions that are presumably involved in transmitting light-induced structural changes which occur in the BLUF domain to the EAL domain. According to the results of simulations and an analysis of the hydrogen bonding between the respective polypeptide chains, the region containing the site on the α3α4 loop of BLUF is responsible for communication between the photosensing and catalytic domains in the dimeric BlrP1 protein.