Real-time tracking of protein unfolding with time-resolved x-ray solution scattering.

The correct folding of proteins is of paramount importance for their function, and protein misfolding is believed to be the primary cause of a wide range of diseases. Protein folding has been investigated with time-averaged methods and time-resolved spectroscopy, but observing the structural dynamics of the unfolding process in real-time is challenging. Here, we demonstrate an approach to directly reveal the structural changes in the unfolding reaction. We use nano- to millisecond time-resolved x-ray solution scattering to probe the unfolding of apomyoglobin. The unfolding reaction was triggered using a temperature jump, which was induced by a nanosecond laser pulse. We demonstrate a new strategy to interpret time-resolved x-ray solution scattering data, which evaluates ensembles of structures obtained from molecular dynamics simulations. We find that apomyoglobin passes three states when unfolding, which we characterize as native, molten globule, and unfolded. The molten globule dominates the population under the conditions investigated herein, whereas native and unfolded structures primarily contribute before the laser jump and 30 µs after it, respectively. The molten globule retains much of the native structure but shows a dynamic pattern of inter-residue contacts. Our study demonstrates a new strategy to directly observe structural changes over the cause of the unfolding reaction, providing time- and spatially resolved atomic details of the folding mechanism of globular proteins.

New Light on the Mechanism of Phototransduction in Phototropin.

Phototropins are photoreceptor proteins that regulate blue light-dependent biological processes for efficient photosynthesis in plants and algae. The proteins consist of a photosensory domain that responds to the ambient light and an output module that triggers cellular responses. The photosensory domain of phototropin from Chlamydomonas reinhardtii contains two conserved LOV (light-oxygen-voltage) domains with flavin chromophores. Blue light triggers the formation of a covalent cysteine-flavin adduct and upregulates the phototropin kinase activity. Little is known about the structural mechanism that leads to kinase activation and how the two LOV domains contribute to this. Here, we investigate the role of the LOV1 domain from C. reinhardtii phototropin by characterizing the structural changes occurring after blue light illumination with nano- to millisecond time-resolved X-ray solution scattering. By structurally fitting the data with atomic models generated by molecular dynamics simulations, we find that adduct formation induces a rearrangement of the hydrogen bond network from the buried chromophore to the protein surface. In particular, the change in conformation and the associated hydrogen bonding of the conserved glutamine 120 induce a global movement of the ß-sheet, ultimately driving a change in the electrostatic potential on the protein surface. On the basis of the change in the electrostatics, we propose a structural model of how LOV1 and LOV2 domains interact and regulate the full-length phototropin from C. reinhardtii. This provides a rationale for how LOV photosensor proteins function and contributes to the optimal design of optogenetic tools based on LOV domains.

[The potential of the non-pharmacological methods for the rehabilitation and prophylaxis in the patients suffering from with atopic dermatitis]. / Vozmozhnosti reabilitatsii bol'nykh atopicheskim dermatitom i profilaktiki obostrenii nelekarstvennymi metodami.

This article was designed to discuss the therapeutic potential of various non-pharmacological and physiotherapeutic methods for the treatment and rehabilitation of the patients presenting with atopic dermatitis (AD) during the inter-recurrence period of the disease. The particular emphasis is placed on the physical agents most frequently used for the purpose with special reference to the combined therapy of atopic dermatitis in the adults and children and to their rehabilitation in the inter-exacerbation periods. In addition, the data on the prospects for the use of various medications intended for tissue- and organotherapy of the patients suffering from atopic dermatitis are presented. The main traditional approaches to the management of the patients with atopic dermatitis under conditions of the spa and health resort facilities are considered based on the original experience of the authors including the application of various modes of ozone therapy regarded as a physiotherapeutic procedure for the treatment of atopic dermatitis in the children and adult patients, their rehabilitation, and the prevention of exacerbations of the pathological process based on the external and/or systemic application of the ozone-oxygen gaseous mixture. The selected modalities of ozone therapy used to treat various clinical forms and stages of the atopic dermatitis differing in severity are described in detail. The data on the influence of ozone therapy on a variety of pathogenetic factors of atopic dermatitis are presented as obtained by the investigations into dynamics of the characteristics of immunity, microcirculation, and the levels of free radical metabolites. The results of the study give evidence of the high effectiveness of ozone therapy as a method of physiotherapeutic treatment both in the capacity of a component of combined therapy during the acute phase of atopic dermatitis and as the means of secondary (post-exposure) prophylaxis of the exacerbations and relapses of this condition based at the medical and preventive treatment facilities of various specialization.

Epiphytic microorganisms degrading aromatic hydrocarbons from the phyllosphere of urban woody plants.

From the leaves of three urban trees (Tilia sp., Acer sp., and Fraxinus sp.), 180 strains degrading phenanthrene, naphthalene, and salicylate were isolated by direct plating and enrichment cultures. The leaves of each tree species were characterized by a specific profile of aromatic hydrocarbon-degrading microflora. Members of the type Actinobacteria were predominant in the case of direct plating on media with phenanthrene and naphthalene. Enrichment cultures with phenanthrene and salicylate were shown to yield microbial consortia, the composition of which changed with time. Members of the type Proteobacteria were predominant in these consortia. No plasmids of polycyclic aromatic hydrocarbon degradation of the P-7 and P-9 incompatibility groups were revealed in the studied strains.

[Occurrence of the SAL+ phenotype in soil pseudomonads].

Genetic systems of salicylate catabolism were studied in 75 strains of fluorescent pseudomoriads and in 30 exogenously isolated SAL plasmids. All exogenously isolated SAL plasmids were found to contain the classical nahG gene in combination with the genes of the meta-pathway of catechol cleavage. In most studied strains, salicylate catabolism was controlled by the chromosomal genes, the nah Ugene being the key gene ofsalicylate utilization and subsequent catechol cleavage occurring via the ortho-pathway. It is suggested that the nah U-like sequences play a key role in occurrence of the Sal+ phenotype in strains degrading salicylate, but not naphthalene.

[Organization and maintenance features of IncP-7 naphthalene degradation plasmid pFME5 basic replicon].

A basic replicon of the naphthalene degradation plasmid pFME5 (80 kb, IncP-7) has been constructed and sequenced. The nucleotide sequence of pFME5mini is almost identical to replicons of the pND6-1 subgroup, which was separated based on the reA-oriV homology in our previous work. The basic replicon of pFME5 is capable of replication and stable maintenance exclusively in Pseudomonas species. An analysis of the deletion mutation indicated that, in contrast to the parWAB region, the parC gene is not essential for the stability of pFME5mini and can be a common feature of IncP-7 replicons. We revealed that par-defective mutants of pFME5mini were slowly eliminated from the bacterial population in a nonselective medium compared to their pCAR1-based counterparts. Designed primers specific to the repA and parC genes can be used to detect IncP-7 plasmids, while primers specific to two variants of parA can be used for intragroup classification.

[IncP-7 plasmids' classification based on structural diversity of their basic replicons].

The structural diversity of basic replicons and repB gene of the IncP-7 plasmids' collection was firstly assessed on the basis of PCR, restriction analysis and partial sequencing. It has been revealed that DNA fragment containing gene for UvrD-like helicase RepB is a part of all known P-7 replicons, but often serves as hot place for diverse IS-elements invasion. The first system of P-7 plasmids' classification has been worked out on the basis of determined repA-oriV-par WABC nucleotide divergency. Most degradation plasmids established to be belonging to large beta-subgroup, streptomycin resistance plasmid Rms148 (IncP-7 archetype)--to alpha-subgroup, carbazole degradation plasmid pCAR1 and NAH/SAL-plasmids from pY-line (Yamal oil deposits)--to gamma-subgroup and CAP-plasmid pBS270 with potentially reduced P-7 replicon--to delta-subgroup. It has been observed that the type of IncP-7 basic replicon molecular organization does not correlate with fixed phenotypic character in most cases, that is plasmids encoding different phenotypic markers could be members of the same P-7 subgroup.

[The new gene encoding TrfA-type replication initiator has been found on caprolactam/salicylate degradation plasmid pBS270].

The mini-replicon of pseudomonads' caprolactam/salicylate degradation plasmid pBS270 (105 kb, contains incompatibility determinants of P-7 group) has been obtained and its nucleotide sequence has been determined. The new gene encoding TrfA-like replication initiator has been found on this replicon. Poor homology of this replication initiator with known proteins of TrfA-family allows us to classify obtained replicon as IncP-1-like. The pBS270mini reveals chimeric nature.

[scpA the new salicylate hydroxylase gene localized on salicylate/caprolactam degradation plasmids].

Both caprolactam and salicylate biodegradation by Pseudomonas salicylate/caprolactam degraders is controlled by large conjugative plasmids (SAL/CAP). Some of these plasmids determined to be the members of IncP-7 group. The new salicylate 1-hydroxylase gene (scpA) on SAL/CAP-plasmids has been detected and partially sequenced. Gene scpA was equally related to closest homologs nahG (NAH7), salA (P. reinekei MT1) and nahU (pND6-1), but identity of scpA to these genes did not exceed 72-74%. Synthesis of salicylate 1-hydroxylase ScpA was not induced by salicylate. This enzyme had wide substrate specificity and exhibited highest specific activity with 4-methylsalicylate and nonsubstituted salicylate. Besides pseudomonad's salicylate degradative conjugative plasmids without "classical" nah2-operon and harboring only salicylate 1-hydroxylase gene nahU have been firstly described.

[Phylogenetic analysis of the genes for naphthalene and phenanthrene degradation in Burkholderia sp. strains].

The genetic systems responsible for naphthalene and phenanthrene catabolism have been analyzed in the five strains of Burkholderia sp. isolated from soil samples (West Siberia) contaminated by heavy residual fuel oil and in the strain Burkholderia sp. BS3702 from the laboratory collection isolated from soil samples of the coke gas works (Vidnoe, Moscow oblast). The results of this work demonstrate that naphthalene and phenanthrene degradation in the above strains is encoded by the sequences not homologous to the classical nah genes of pseudomonades. In the Burkholderia sp. BS3702 strain, the initial stages of phenanthrene degradation and the subsequent stages of salicylate degradation are controlled by the sequences of different evolutionary origins (phn and nag genes).

[The structure of replication initiation region of Pseudomonas IncP-7 streptomycin resistance plasmid Rms148].

Pseudomonads' IncP-7 plasmids make significant contribution to the environmental biodegradative potential and sometimes harbour antibiotic resistance genes. More than 30 years plasmid Rms148 is used as archetypal P-7 plasmid in microbiological incompatibility tests. However, the structure of its basic replicon was not described up to now, as well as phylogenetic relationships between all known plasmids within the IncP-7 group were not studied. In the frames of this work we have constructed two primer pairs to amplify main components of P-7 replication initiation region, and subsequent screening of repA intragenic polymorphism was made using laboratory collection of IncP-7 plasmids. Minimal replicon of Rms148 was constructed and its nucleotide sequence was determined to be identical to repA-oriVof known P-7 plasmids on 81-83% and forming separate branch on appropriate phylogenetic tree. Additionally, repA seems to be more conservative between group members compared with putative oriV region. Deduced amino acid sequence and predicted secondary and tertiary structures of Rms148 RepA protein allow us to make assumption about similar to unclassified cryptic plasmid pPS10 model of replication initiation for IncP-7 group members.

BioCARS: a synchrotron resource for time-resolved X-ray science.

BioCARS, a NIH-supported national user facility for macromolecular time-resolved X-ray crystallography at the Advanced Photon Source (APS), has recently completed commissioning of an upgraded undulator-based beamline optimized for single-shot laser-pump X-ray-probe measurements with time resolution as short as 100 ps. The source consists of two in-line undulators with periods of 23 and 27 mm that together provide high-flux pink-beam capability at 12 keV as well as first-harmonic coverage from 6.8 to 19 keV. A high-heat-load chopper reduces the average power load on downstream components, thereby preserving the surface figure of a Kirkpatrick-Baez mirror system capable of focusing the X-ray beam to a spot size of 90 µm horizontal by 20 µm vertical. A high-speed chopper isolates single X-ray pulses at 1 kHz in both hybrid and 24-bunch modes of the APS storage ring. In hybrid mode each isolated X-ray pulse delivers up to ~4 × 10(10) photons to the sample, thereby achieving a time-averaged flux approaching that of fourth-generation X-FEL sources. A new high-power picosecond laser system delivers pulses tunable over the wavelength range 450-2000 nm. These pulses are synchronized to the storage-ring RF clock with long-term stability better than 10 ps RMS. Monochromatic experimental capability with Biosafety Level 3 certification has been retained.

[Horizontal transfer of catabolic plasmids in the process of naphthalene biodegradation in model soil systems].

The process of naphthalene degradation by indigenous, introduced, and transconjugant strains was studied in laboratory soil microcosms. Conjugation transfer of catabolic plasmids was demonstrated in naphthalene-contaminated soil. Both indigenous microorganisms and an introduced laboratory strain BS394 (pNF142::TnMod-OTc) served as donors of these plasmids. The indigenous bacterial degraders of naphthalene isolated from soil were identified as Pseudomonas putida and Pseudomonas fluorescens. The frequency of plasmid transfer in soil was 10(-5)-10(-4) per donor cell. The activity of the key enzymes of naphthalene biodegradation in indigenous and transconjugant strains was studied. Transconjugant strains harboring indigenous catabolic plasmids possessed high salicylate hydroxylase and low catechol-2,3-dioxygenase activities, in contrast to indigenous degraders, which had a high level of catechol-2,3-dioxygenase activity and a low level of salicylate hydroxylase. Naphthalene degradation in batch culture in liquid mineral medium was shown to accelerate due to cooperation of the indigenous naphthalene degrader P. fluorescens AP1 and the transconjugant strain P. putida KT2442 harboring the indigenous catabolic plasmid pAP35. The role of conjugative transfer of naphthalene biodegradation plasmids in acceleration of naphthalene degradation was demonstrated in laboratory soil microcosms.

[Salicylate degradation by Pseudomonas putida strains not involving the "classical" nah2 operon].

Genetic systems for salicylate catabolism were analyzed in 12 strains of Pseudomonas putida, isolated from polluted soil samples collected in the Murmansk and Tula oblasts. All of the studied P. putida strains utilize salicylate in the ortho-pathway of catechol cleavage without employing the enzymes of the "classical" nah2 operon. The data demonstrates that salicylate degradation in the studied strains is performed with the involvement of the salicylate hydroxylase gene analogous to the nahU gene of strain P. putida ND6. New variants of salicylate hydroxylase genes nahG1 and nahU were found.

[Nah-plasmids of IncP-9 group from natural strains of Pseudomonas].

Use of polymerase chain reaction helped to establish that the most frequent among naphthalene utilizing bacteria, isolated on the territory of Belarus, are Nah-plasmids of IncP-9 incompatibility group and those with indefinite systematic belonging. With the help of classical test of incompatibility, restriction and sequence analyses three new subgroups within the IncP-9 group were discovered (zeta, eta and IncP-9-like replicons). Conducting of restriction analysis for amplification products of nahG and nahAc genes allowed us to reveal, in addition to known sequences of stated determinants, two new types of nahG gene. Restriction analysis performed on amplification products of 16S RNA genes (ARDRA method) showed that native hosts of Nah-plasmids of IncP-9 group are not only fluorescent bacteria from genus Pseudomonas (P. fluorescens, P. putida, P. aeruginosa, P. species), but also non-fluorescent bacteria with indefinite specific belonging.

[The construction and monitoring of genetically marked, plasmid-containing, naphthalene-degrading strains in soil].

A genetically marked, plasmid-containing, naphthalene-degrading strain, Pseudomonas putida KT2442(pNF142::TnMod-OTc), has been constructed. The presence of the gfp gene (which codes for green fluorescent protein) and the kanamycin and rifampicin resistance genes in the chromosome of this strain allows the strain's fate in model soil systems to be monitored, whereas a minitransposon, built in naphthalene biodegradation plasmid pNF142, contains the tetracycline resistance gene and makes it possible to follow the horizontal transfer of this plasmid between various bacteria. Plasmid pNF142::TnMod-OTc is stable in strain P. putida KT2442 under nonselective conditions. The maximal specific growth rate of this strain on naphthalene was found to be higher than that of the natural host of plasmid pNF142. When introduced into a model soil system, the genetically marked strain is stable and competitive for 40 days. The transfer of marked plasmid pNF142::TnMod-OTc to natural soil bacteria, predominantly fluorescent pseudomonads, has been detected.

[Incompatibility group P-7 plasmids responsible for biodegradation of naphthalene and salicylate in fluorescent pseudomonads].

Analysis of seven plasmids (77 to 135 kbp in size) of the P-7 incompatibility group that are responsible for the biodegradation of naphthalene and salicylate has shown that the main natural host of IncP-7 plasmids is the species Pseudomonas fluorescens. The IncP-7 plasmids are structurally diverse and do not form groups, as is evident from their cluster analysis. The naphthalene catabolism genes of six of the IncP-7 plasmids are conservative and homologous to the catabolic genes of NAH7 and pDTG1 plasmids. The pAK5 plasmid contains the classical nahA gene, which codes for naphthalene dioxygenase, and the salicylate 5-hydroxylase gene (nagG) sequence, which makes the conversion of salicylate to gentisate possible.