Expression, Purification and Characterization of a ZIP Family Transporter from Desulfovibrio vulgaris.

The ZIP family transport zinc ions from the extracellular medium across the plasma membrane or from the intracellular compartments across endomembranes, which play fundamental roles in metal homeostasis and are broadly involved in physiological and pathological processes. Desulfovibrio is the predominant sulphate-reducing bacteria in human colonic microbiota, but also a potential choice for metal bioremediation. while, there are no published studies describing the zinc transporters from Desulfovibrio up to now. In this study, we obtained for the first time a heterologously expressed ZIP homolog from Desulfovibrio vulgaris, termed dvZip. The purified dvZip was reconstituted into proteoliposomes, and confirmed its zinc transport ability in vitro. By combining topology prediction, homology modeling and phylogenetic approaches, we also noticed that dvZip belongs to the GufA and probably have 8 transmembrane α-helical segments (TM 1-8) in which both termini are located on the extracellular, with TM2, 4, 5 and 7 create an inner bundle. We believe that purification and characterization of zinc (probably also cadmium) transporters from Desulfovibrio vulgaris such as dvZip could shed light on understanding of metal homeostasis of Desulfovibrio and provided protein products for future detailed function and structural studies.

Computational approaches to amino acid side-chain conformation using combined NMR theoretical and experimental results: leucine-67 in Desulfovibrio vulgaris flavodoxin.

In this paper, we show that the combination of NMR theoretical and experimental results can help to solve the molecular structure of peptides, here it is used as an example the residue Leucine-67 in Desulfovibrio vulgaris flavodoxin. We apply a computational protocol based on the leucine amino acid dipeptide, which, using calculated and experimental spin-spin coupling constants, allows us to obtain the conformation of the amino acid side chain. Calculated results show that the best agreement is obtained when three conformers around the lateral chain angle $\chi _1$ are considered or when the dynamic effect in the torsional angles is included. The population of each structure is estimated and analyzed according to the correlation between those two approaches. Independently of the approach, the estimated $\chi _1$ angle in solution is close to the staggered value of -60$^\circ $ and deviates significantly from the average x-ray angle of -90$^\circ $.

Structural basis for assembly of non-canonical small subunits into type I-C Cascade.

Bacteria and archaea employ CRISPR (clustered, regularly, interspaced, short palindromic repeats)-Cas (CRISPR-associated) systems as a type of adaptive immunity to target and degrade foreign nucleic acids. While a myriad of CRISPR-Cas systems have been identified to date, type I-C is one of the most commonly found subtypes in nature. Interestingly, the type I-C system employs a minimal Cascade effector complex, which encodes only three unique subunits in its operon. Here, we present a 3.1 Å resolution cryo-EM structure of the Desulfovibrio vulgaris type I-C Cascade, revealing the molecular mechanisms that underlie RNA-directed complex assembly. We demonstrate how this minimal Cascade utilizes previously overlooked, non-canonical small subunits to stabilize R-loop formation. Furthermore, we describe putative PAM and Cas3 binding sites. These findings provide the structural basis for harnessing the type I-C Cascade as a genome-engineering tool.

Mechanism and Application of the Catalytic Reaction of [NiFe] Hydrogenase: Recent Developments.

Hydrogenases (H2 ase) catalyze the oxidation of dihydrogen and the reduction of protons with remarkable efficiency, thereby attracting considerable attention in the energy field due to their biotechnological potential. For this simple reaction, [NiFe] H2 ase has developed a sophisticated but intricate mechanism with the heterolytic cleavage of dihydrogen, where its Ni-Fe active site exhibits various redox states. Recently, new spectroscopic and crystal structure studies of [NiFe] H2 ases have been reported, providing significant insights into the catalytic reaction mechanism, hydrophobic gas-access tunnel, proton-transfer pathway, and electron-transfer pathway of [NiFe] H2 ases. In addition, [NiFe] H2 ases have been shown to play an important role in biofuel cell and solar dihydrogen production. This concept provides an overview of the biocatalytic reaction mechanism and biochemical application of [NiFe] H2 ases based on the new findings.

Suppressing hydrogen peroxide generation to achieve oxygen-insensitivity of a [NiFe] hydrogenase in redox active films.

Redox-active films were proposed as protective matrices for preventing oxidative deactivation of oxygen-sensitive catalysts such as hydrogenases for their use in fuel cells. However, the theoretical models predict quasi-infinite protection from oxygen and the aerobic half-life for hydrogenase-catalyzed hydrogen oxidation within redox films lasts only about a day. Here, we employ operando confocal microscopy to elucidate the deactivation processes. The hydrogen peroxide generated from incomplete reduction of oxygen induces the decomposition of the redox matrix rather than deactivation of the biocatalyst. We show that efficient dismutation of hydrogen peroxide by iodide extends the aerobic half-life of the catalytic film containing an oxygen-sensitive [NiFe] hydrogenase to over one week, approaching the experimental anaerobic half-life. Altogether, our data support the theory that redox films make the hydrogenases immune against the direct deactivation by oxygen and highlight the importance of suppressing hydrogen peroxide production in order to reach complete protection from oxidative stress.

Postnovo: Postprocessing Enables Accurate and FDR-Controlled de Novo Peptide Sequencing.

De novo sequencing offers an alternative to database search methods for peptide identification from mass spectra. Since it does not rely on a predetermined database of expected or potential sequences in the sample, de novo sequencing is particularly appropriate for samples lacking a well-defined or comprehensive reference database. However, the low accuracy of many de novo sequence predictions has prevented the widespread use of the variety of sequencing tools currently available. Here, we present a new open-source tool, Postnovo, that postprocesses de novo sequence predictions to find high-accuracy results. Postnovo uses a predictive model to rescore and rerank candidate sequences in a manner akin to database search postprocessing tools such as Percolator. Postnovo leverages the output from multiple de novo sequencing tools in its own analyses, producing many times the length of amino acid sequence information (including both full- and partial-length peptide sequences) at an equivalent false discovery rate (FDR) compared to any individual tool. We present a methodology to reliably screen the sequence predictions to a desired FDR given the Postnovo sequence score. We validate Postnovo with multiple data sets and demonstrate its ability to identify proteins that are missed by database search even in samples with paired reference databases.

Reactivation of standard [NiFe]-hydrogenase and bioelectrochemical catalysis of proton reduction and hydrogen oxidation in a mediated-electron-transfer system.

Standard [NiFe]-hydrogenase from Desulfovibrio vulgaris Miyazaki F (DvMF-H2ase) catalyzes the uptake and production of hydrogen (H2) and is a promising biocatalyst for future energy devices. However, DvMF-H2ase experiences oxidative inactivation under oxidative stress to generate Ni-A and Ni-B states. It takes a long time to reactivate the Ni-A state by chemical reduction, whereas the Ni-B state is quickly reactivated under reducing conditions. Oxidative inhibition limits the application of DvMF-H2ase in practical devices. In this research, we constructed a mediated-electron-transfer system by co-immobilizing DvMF-H2ase and a viologen redox polymer (VP) on electrodes. The system can avoid oxidative inactivation into the Ni-B state at high electrode potentials and rapidly reactivate the Ni-A state by electrochemical reduction of VP. H2 oxidation and H+ reduction were realized by adjusting the pH from a thermodynamic viewpoint. Using carbon felt as a working-electrode material, high current densities-up to (200 ±â€¯70) and -(100 ±â€¯9) mA cm-3 for the H2-oxidation and H+-reduction reactions, respectively-were attained.

Conformational difference between two subunits in flavin mononucleotide binding protein dimers from Desulfovibrio vulgaris (MF): molecular dynamics simulation.

The structural and dynamical properties of five FMN binding protein (FBP) dimers, WT (wild type), E13K (Glu13 replaced by Lys), E13R (Glu13 replaced by Arg), E13T (Glu13 replaced by Thr) and E13Q (Glu13 replaced by Gln), were investigated using a method of molecular dynamics simulation (MDS). In crystal structures, subunit A (Sub A) and subunit B (Sub B) were almost completely equivalent in all of the five FBP dimers. However, the predicted MDS structures of the two subunits were not equivalent in solution, revealed by the distances and inter-planar angles between isoalloxazine (Iso) and aromatic amino acids (Trp32, Tyr35 and Trp106) as well as the hydrogen bonding pairs between Iso and nearby amino acids. Residue root of mean square fluctuations (RMSF) also displayed considerable differences between Sub A and Sub B and in the five FBP dimers. The dynamics of the whole protein structures were examined with the distance (RNN) between the peptide N atom of the N terminal (Met1) and the peptide N atom of the C terminal (Leu122). Water molecules were rarely accessible to Iso in all FBP dimers which are in contrast with other flavoenzymes.

Comparative analysis of the mechanisms of sulfur anion oxidation and reduction by dsr operon to maintain environmental sulfur balance.

Sulfur metabolism is one of the oldest known redox geochemical cycles in our atmosphere. These redox processes utilize different sulfur anions and the reactions are performed by the gene products of dsr operon from phylogenetically diverse sets of microorganisms. The operon is involved in the maintenance of environmental sulfur balance. Interestingly, the dsr operon is found to be present in both sulfur anion oxidizing and reducing microorganisms and in both types of organisms DsrAB protein complex plays a vital role. Though there are various reports regarding the genetics of dsr operon there are practically no reports dealing with the structural aspects of sulfur metabolism by dsr operon. In our present study, we tried to compare the mechanisms of sulfur anion oxidation and reduction by Allochromatium vinosum and Desulfovibrio vulgaris respectively through DsrAB protein complex. We analyzed the modes of bindings of sulfur anions to the DsrAB protein complex and observed that for sulfur anion oxidizers, sulfide and thiosulfate are the best substrates whereas for reducers sulfate and sulfite have the best binding abilities. We analyzed the binding interaction pattern of the DsrA and DsrB proteins while forming the DsrAB protein complexes in Desulfovibrio vulgaris and Allochromatium vinosum. To our knowledge this is the first report that analyzes the differences in binding patterns of sulfur substrates with DsrAB protein from these two microorganisms. This study would therefore be essential to predict the biochemical mechanism of sulfur anion oxidation and reduction by these two microorganisms i.e., Desulfovibrio vulgaris (sulfur anion reducer) and Allochromatium vinosum (sulfur anion oxidizer). Our observations also highlight the mechanism of sulfur geochemical cycle which has important implications in future study of sulfur metabolism as it has a huge application in waste remediation and production of industrial bio-products viz. vitamins, bio-polyesters and bio-hydrogen.

DNA-affinity-purified chip (DAP-chip) method to determine gene targets for bacterial two component regulatory systems.

In vivo methods such as ChIP-chip are well-established techniques used to determine global gene targets for transcription factors. However, they are of limited use in exploring bacterial two component regulatory systems with uncharacterized activation conditions. Such systems regulate transcription only when activated in the presence of unique signals. Since these signals are often unknown, the in vitro microarray based method described in this video article can be used to determine gene targets and binding sites for response regulators. This DNA-affinity-purified-chip method may be used for any purified regulator in any organism with a sequenced genome. The protocol involves allowing the purified tagged protein to bind to sheared genomic DNA and then affinity purifying the protein-bound DNA, followed by fluorescent labeling of the DNA and hybridization to a custom tiling array. Preceding steps that may be used to optimize the assay for specific regulators are also described. The peaks generated by the array data analysis are used to predict binding site motifs, which are then experimentally validated. The motif predictions can be further used to determine gene targets of orthologous response regulators in closely related species. We demonstrate the applicability of this method by determining the gene targets and binding site motifs and thus predicting the function for a sigma54-dependent response regulator DVU3023 in the environmental bacterium Desulfovibrio vulgaris Hildenborough.

Automated particle correspondence and accurate tilt-axis detection in tilted-image pairs.

Tilted electron microscope images are routinely collected for an ab initio structure reconstruction as a part of the Random Conical Tilt (RCT) or Orthogonal Tilt Reconstruction (OTR) methods, as well as for various applications using the "free-hand" procedure. These procedures all require identification of particle pairs in two corresponding images as well as accurate estimation of the tilt-axis used to rotate the electron microscope (EM) grid. Here we present a computational approach, PCT (particle correspondence from tilted pairs), based on tilt-invariant context and projection matching that addresses both problems. The method benefits from treating the two problems as a single optimization task. It automatically finds corresponding particle pairs and accurately computes tilt-axis direction even in the cases when EM grid is not perfectly planar.

Photoinduced electron transfer modeling to simulate flavoprotein fluorescence decay.

A method of analysis is described on the photoinduced electron transfer (PET) from aromatic amino acids as tryptophans (Trp) and tyrosines (Tyr) to the excited isoalloxazine (Iso*) in FMN-binding proteins (FBP) from Desulfovibrio vulgaris (strain, Miyazaki F). Time-dependent geometrical factors as the donor-acceptor distances are determined by means of a molecular dynamics simulation (MDS) of the proteins. Fluorescence decays of the single mutated isoforms of FBP are used as experimental data. The electrostatic (ES) energy between the photoproducts and ionic groups in the proteins is introduced into the Kakitani and Mataga (KM) model, which is modeled for an electron transfer process in solution. The PET parameters contained in the KM rate are determined by means of a nonlinear least square method, according to the Marquardt algorithm. The agreement between the observed and calculated decays is quite good, but not optimal. Characteristics on PET in flavoproteins, obtained by the present method, are described. Possible improvements of the method are discussed.

Effect of the protonation degree of a self-assembled monolayer on the immobilization dynamics of a [NiFe] hydrogenase.

Understanding the interaction and immobilization of [NiFe] hydrogenases on functionalized surfaces is important in the field of biotechnology and, in particular, for the development of biofuel cells. In this study, we investigated the adsorption behavior of the standard [NiFe] hydrogenase of Desulfovibrio gigas on amino-terminated alkanethiol self-assembled monolayers (SAMs) with different levels of protonation. Classical all-atom molecular dynamics (MD) simulations revealed a strong correlation between the adsorption behavior and the level of ionization of the chemically modified electrode surface. While the hydrogenase undergoes a weak but stable initial adsorption process on SAMs with a low degree of protonation, a stronger immobilization is observable on highly ionized SAMs, affecting protein reorientation and conformation. These results were validated by complementary surface-enhanced infrared absorption (SEIRA) measurements on the comparable [NiFe] standard hydrogenases from Desulfovibrio vulgaris Miyazaki F and allowed in this way for a detailed insight into the adsorption mechanism at the atomic level.

Charge parametrization of the DvH-c3 heme group: validation using constant-(pH,E) molecular dynamics simulations.

We studied the effect of using different heme group charge parametrization methods and schemes (Merz-Kollman, CHELPG, and single- and multiconformational RESP) on the quality of the results produced by the constant-(pH,E) MD method, applied to the redox titration of Desulfovibrio vulgaris Hildenborough cytochrome c(3). These new and more accurate charge sets enabled us to overcome the previously reported dependence of the method's performance on the dielectric constant, ε, assigned to the protein region. In particular, we found a systematic, clear shift of the E(mod) toward more negative values than those previously reported, in agreement with an electrostatics based reasoning. The simulations showed strong coupling between protonating/redox sites. We were also able to capture significant direct and, especially, indirect interactions between hemes, such as those mediated by histidine 67. Our results highlight the importance of having a good quantum description of the system before deriving atomic partial charges for classic force fields.

High-throughput isolation and characterization of untagged membrane protein complexes: outer membrane complexes of Desulfovibrio vulgaris.

Cell membranes represent the "front line" of cellular defense and the interface between a cell and its environment. To determine the range of proteins and protein complexes that are present in the cell membranes of a target organism, we have utilized a "tagless" process for the system-wide isolation and identification of native membrane protein complexes. As an initial subject for study, we have chosen the Gram-negative sulfate-reducing bacterium Desulfovibrio vulgaris. With this tagless methodology, we have identified about two-thirds of the outer membrane- associated proteins anticipated. Approximately three-fourths of these appear to form homomeric complexes. Statistical and machine-learning methods used to analyze data compiled over multiple experiments revealed networks of additional protein-protein interactions providing insight into heteromeric contacts made between proteins across this region of the cell. Taken together, these results establish a D. vulgaris outer membrane protein data set that will be essential for the detection and characterization of environment-driven changes in the outer membrane proteome and in the modeling of stress response pathways. The workflow utilized here should be effective for the global characterization of membrane protein complexes in a wide range of organisms.

Distant residues mediate picomolar binding affinity of a protein cofactor.

Numerous proteins require cofactors to be active. Computer simulations suggest that cooperative interaction networks achieve optimal cofactor binding. There is a need for the experimental identification of the residues crucial for stabilizing these networks and thus for cofactor binding. Here we investigate the electron transporter flavodoxin, which contains flavin mononucleotide as non-covalently bound cofactor. We show that after binding flavin mononucleotide with nanomolar affinity, the protein relaxes extremely slowly (time constant ~5 days) to an energetically more favourable state with picomolar-binding affinity. Rare small-scale openings of this state are revealed through H/D exchange of N(3)H of flavin. We find that H/D exchange can pinpoint amino acids that cause tight cofactor binding. These hitherto unknown residues are dispersed throughout the structure, and many are located distantly from the flavin and seem irrelevant to flavodoxin's function. Quantification of the thermodynamics of ligand binding is important for understanding, engineering, designing and evolving ligand-binding proteins.

Effects of high CO2 concentrations on ecophysiologically different microorganisms.

We investigated the effect of increasing CO(2) concentrations on the growth and viability of ecophysiologically different microorganisms to obtain information for a leakage scenario of CO(2) into shallow aquifers related to the capture and storage of CO(2) in deep geological sections. CO(2) concentrations in the gas phase varied between atmospheric conditions and 80% CO(2) for the aerobic strains Pseudomonas putida F1 and Bacillus subtilis 168 and up to 100% CO(2) for the anaerobic strains Thauera aromatica K172 and Desulfovibrio vulgaris Hildenborough. Increased CO(2) concentrations caused prolonged lag-phases, and reduced growth rates and cell yields; the extent of this effect was proportional to the CO(2) concentration. Additional experiments with increasing CO(2) concentrations and increasing pressure (1-5000 kPa) simulated situations occurring in deep CO(2) storage sites. Living cell numbers decreased significantly within 24 h at pressures ≥1000 kPa, demonstrating a severe lethal effect for the combination of high pressure and CO(2).

Solution NMR structure, backbone dynamics, and heme-binding properties of a novel cytochrome c maturation protein CcmE from Desulfovibrio vulgaris.

Cytochrome c maturation protein E, CcmE, plays an integral role in the transfer of heme to apocytochrome c in many prokaryotes and some mitochondria. A novel subclass featuring a heme-binding cysteine has been identified in archaea and some bacteria. Here we describe the solution NMR structure, backbone dynamics, and heme binding properties of the soluble C-terminal domain of Desulfovibrio vulgaris CcmE, dvCcmE'. The structure adopts a conserved ß-barrel OB fold followed by an unstructured C-terminal tail encompassing the CxxxY heme-binding motif. Heme binding analyses of wild-type and mutant dvCcmE' demonstrate the absolute requirement of residue C127 for noncovalent heme binding in vitro.

Crystal structure and spectroscopic studies of a stable mixed-valent state of the hemerythrin-like domain of a bacterial chemotaxis protein.

The bacterial chemotaxis protein of Desulfovibrio vulgaris DcrH (DcrH-Hr) functions as an O(2)-sensing protein. This protein has a hemerythrin-like domain that includes a nonheme diiron center analogous to the diiron center of the hemerythrin (Hr) family. Interestingly, the O(2) affinity of DcrH-Hr is 3.3 × 10(6) M(-1), a value 25-fold higher than that of the Pectinaria gouldii Hr. This high affinity arises from the fast association of the O(2) ligand with DcrH-Hr (k(on) = 5.3 × 10(8) M(-1) s(-1)), which is made possible by a hydrophobic tunnel that accelerates the passage of the O(2) ligand to the diiron site. Furthermore, the autoxidation kinetics indicate that the rate of autoxidation of DcrH-Hr is 54-fold higher than that of P. gouldii Hr, indicating that the oxy form of DcrH-Hr is not stable toward autoxidation. More importantly, a mixed-valent state, semimet(R), which was spectroscopically observed in previous Hr studies, was found to be stable for over 1 week and isolable in the case of DcrH-Hr. The high-resolution crystal structures of the semimet(R)- (1.8 Å) and met-DcrH-Hr (1.4 Å) indicate that the semimet(R)- and met-DcrH-Hr species have very similar coordination geometry at the diiron site.

Communication: Accurate determination of side-chain torsion angle χ1 in proteins: phenylalanine residues.

Quantitative side-chain torsion angle χ(1) determinations of phenylalanine residues in Desulfovibrio vulgaris flavodoxin are carried out using exclusively the correlation between the experimental vicinal coupling constants and theoretically determined Karplus equations. Karplus coefficients for nine vicinal coupling related with the torsion angle χ(1) were calculated using the B3LYP functional and basis sets of different size. Optimized χ(1) angles are in outstanding agreement with those previously reported by employing x ray and NMR measurements.