Primary Sequence Analysis Research Articles

Exploring the salty peptides of enzymatically hydrolyzed Volvariella volvacea protein: Structural analysis and taste perception insights

This study aimed to prepare and screen salty peptides and elucidate the factors triggering their saltiness properties and saltiness-enhancing effects. Electronic tongue results indicated that samples treated with Alcalase (AJ) exhibited the highest saltiness intensity (5.05 ± 0.11) and greater saltiness-enhancing compared to those treated with Flavourzyme (AF), Neutrase (AZ), and Papain (AM). Peptides identified by LC-MS/MS were docked to the TMC4 receptor, resulting in 23 peptides with good docked results. The key amino acid residues (Thr431, Arg433, Phe440, Phe432, and Ser273) were discovered. The salty peptides WPGFK (633.75 Da), YFDWPGFK (1059.17 Da), and YFDWPGFKT (1160.27 Da) were selected for salt reduction and molecular characterization studies. Electronic tongue results showed that the 0.01 % WPGFK solution matched the saltiness of the 0.24 % NaCl solution and demonstrated superior saltiness-enhancing compared to YFDWPGFK and YFDWPGFKT. Primary sequence analysis of three salty peptides suggested that the WPG amino acid segment might play a crucial role in generating saltiness. Circular dichroism (CD) analysis of the secondary structure indicated that increased random coil and decreased β-turn contents resulted in higher saltiness perception and increased random coil content led to greater saltiness-enhancing ability.

The Crystal Structure of the Domain of Unknown Function1480 (DUF1480) From Klebsiella pneumoniae.

Domains of unknown function (DUFs) continue to comprise a significant portion of bacterial proteomes, with more than 20% of bacterial proteins remaining annotated as DUFs. The characterization of their molecular structure can provide valuable insight that is not captured by the primary sequence analysis, thus providing a segue into the identification of the molecular function of DUF representatives. Here, we present the crystal structure of KPN_02352 from Klebsiella pneumoniae subsp. pneumoniae, a DUF1480 domain-containing protein, which was determined to be 1.75 Å resolution. Representatives of the DUF1480 family are found broadly across Enterobacterales and have been previously shown to contribute to the antibiotic response. Our structural analysis suggests that DUF1480 is comprised of a six-stranded split barrel fold featuring a small alpha helix that is positioned to cap one end of the split barrel. DUF1480 was found to be monomeric in solution, and harbors structural similarity to response regulators. The crystal structure of DUF1480 is the first experimental insight into the molecular structure of this conserved protein family, revealing several conserved features that may be functionally relevant.

In Vitro Selection and Characterization of a Light-up DNA Aptamer for Thiazole Orange.

A new DNA aptamer that binds to the target Thiazole Orange-biotin (TO1-biotin) was isolated after nine rounds of in vitro selection. The selection was performed on streptavidin-coated beads with the target bound to the surface and with free dye in solution in higher selection rounds to select for slower off-rate binding. Using next-generation sequencing (NGS), the libraries after the 4th and 9th rounds of selection were sequenced to identify enriched sequences. Several sequence families emerged, showing superior fluorescence enhancement and high affinity for the target compared to the other families obtained by NGS analysis. These sequence families were further studied to understand the binding interactions better. Primary sequence and secondary structure analysis tools were used to identify a hypothetical three-tiered G-quadruplex motif for these families. This indicates that the TO1-biotin DNA aptamer identified here uses a similar ligand-binding topology to the original Mango RNA aptamer.

Cancer-Related Mutations in the Sam Domains of EphA2 Receptor and Ship2 Lipid Phosphatase: A Computational Study.

The lipid phosphatase Ship2 interacts with the EphA2 receptor by forming a heterotypic Sam (sterile alpha motif)-Sam complex. Ship2 works as a negative regulator of receptor endocytosis and consequent degradation, and anti-oncogenic effects in cancer cells should be induced by hindering its association with EphA2. Herein, a computational approach is presented to investigate the relationship between Ship2-Sam/EphA2-Sam interaction and cancer onset and further progression. A search was first conducted through the COSMIC (Catalogue of Somatic Mutations in Cancer) database to identify cancer-related missense mutations positioned inside or close to the EphA2-Sam and Ship2-Sam reciprocal binding interfaces. Next, potential differences in the chemical-physical properties of mutant and wild-type Sam domains were evaluated by bioinformatics tools based on analyses of primary sequences. Three-dimensional (3D) structural models of mutated EphA2-Sam and Ship2-Sam domains were built as well and deeply analysed with diverse computational instruments, including molecular dynamics, to classify potentially stabilizing and destabilizing mutations. In the end, the influence of mutations on the EphA2-Sam/Ship2-Sam interaction was studied through docking techniques. This in silico approach contributes to understanding, at the molecular level, the mutation/cancer relationship by predicting if amino acid substitutions could modulate EphA2 receptor endocytosis.

Bioinformatic Analysis of Immunodominant Peptides of Rabies Virus (<i>Rabies lyssavirus, Rhabdoviridae</i>)

There is a need to develop a new generation of anti-rabies vaccines that provide a protective level of antibodies after a single injection. Prospects for solving this problem are opened by the latest developments in the field of “reverse vaccinology”. The main parameter that determines the effectiveness of recombinant vaccines is the design of the antigen-coding sequence. In this regard, the aim of the work was to conduct a bioinformatic analysis of rabies virus (Rabies lyssavirus, Rhabdoviridae) peptides to identify immunogenic epitopes.Materials and methods. Analysis of 5 candidate protein sequences of more than 100 strains and epizootic isolates of the rabies virus was performed using standard in silico prediction methods using Immune Epitope Database (IEDB) (NIH, USA).Results and discussion. As a result of the analysis of primary amino acid sequences, carried out using the most commonly used bioinformatics tools, the number of immunogenic epitopes and the types of immune response detected (T- and B-cell epitopes, class I MHCbinding epitopes) were established for viral proteins: glycoprotein (G), nucleoprotein (N), phosphoprotein (P), matrix protein (M), RNA-dependent RNA polymerase (L). In the amino acid structure of these proteins, N- and O-glycosylation sites, signal peptides, and transmembrane domains were additionally identified. In order to predict the safety and efficacy of these proteins as components of recombinant vaccines, an in silico assessment of their physicochemical properties was carried out. Despite the fact that the predominant number of epitopes is concentrated in the structure of the glycoprotein, the epitopes of other proteins, ranging according to the level of antigenicity and conservatism, may also be of interest as components of preventive drugs or diagnostics. The presented data can be used in the design of the insert during the construction of a candidate virus-vector vaccine or control positive samples in diagnostic methods based on the indication of viral genome fragments.

Cyanobacteria in Hypolithic Horizons of Soils in the Larsemann Hills Oasis, East Antarctica

The research is devoted to the analysis of biodiversity of Antarctic cyanobacteria in hypolithic organic-accumulative horizons of soils in the Larsemann Hills oasis. Studies of fouling glasses by the methods of light and confocal microscopy, as well as by fluorescent in situ hybridization, indicate that filamentous forms predominate among cyanobacteria in the upper layers of Antarctic hypolithic microbial communities. Strains of the genera Nostoc, Halotia, Leptolyngbya, Plectolyngbya, and Phormidesmis, as well as some new and previously undescribed cyanobacteria, were isolated from corresponding soil samples to clarify their taxonomic status. As a result, a unique collection of Antarctic cyanobacteria isolated from soils was obtained for the first time. The strains were described according to the modern polyphasic taxonomy methods based on an integrated assessment of morphological and molecular-genetic features. Phylogenetic analysis of primary 16S rRNA sequences and peculiarities of secondary structures of internal transcribed spacers enabled us to identify new taxa of potentially endemic cyanobacteria among the strains studied. The high level of similarity between the 16S rRNA gene sequences of soil strains and those previously found in benthic mats of water bodies in the Larsemann Hills confirmed the ability of cyanobacteria to expand beyond different ecological niches and to adapt to contrasting environmental conditions.

Structural Analysis of MALAT1 long non-coding RNA in cells and in evolution.

Although not canonically polyadenylated, the long non-coding RNA MALAT1 (Metastasis Associated Lung Adenocarcinoma Transcript 1) is stabilized by a highly conserved 76 nucleotide triple helix structure on its 3' end. The entire MALAT1 transcript is over 8,000 nucleotides long in humans. The strongest structural conservation signal in MALAT1 (as measured by co-variation of base-pairs) is in the triple helix structure. Primary sequence analysis of co-variation alone does not reveal the degree of structural conservation of the entire full-length transcript, however. Furthermore, RNA structure is often context dependent; RNA binding proteins that are differentially expressed in different cell types may alter structure. We investigate here the in cell and cell free structures of the full-length human and green monkey (Chlorocebus sabaeus) MALAT1 transcripts in multiple tissue-derived cell lines using SHAPE chemical probing. Our data reveal levels of uniform structural conservation in different cell lines, in cells and cell free, and even between species, despite significant differences in primary sequence. The uniformity of the structural conservation across the entire transcript suggests that, despite seeing co-variation signals only in the triple helix junction of the lncRNA, the rest of the transcript's structure is remarkably conserved at least in primates and across multiple cell types and conditions.

Secrets behind Protein Sequences: Unveiling the Potential Reasons for Varying Allergenicity Caused by Caseins from Cows, Goats, Camels, and Mares Based on Bioinformatics Analyses.

This study systematically investigated the differences in allergenicity of casein in cow milk (CM), goat milk (GM), camel milk (CAM), and mare milk (MM) from protein structures using bioinformatics. Primary structure sequence analysis reveals high sequence similarity between the α-casein of CM and GM, while all allergenic subtypes are likely to have good hydrophilicity and thermal stability. By analyzing linear B-cell epitope, T-cell epitope, and allergenic peptides, the strongest casein allergenicity is observed for CM, followed by GM, and the casein of MM has the weakest allergenicity. Meanwhile, 7, 9, and 16 similar or identical amino acid fragments in linear B-cell epitopes, T-cell epitopes, and allergenic peptides, respectively, were observed in different milks. Among these, the same T-cell epitope FLGAEVQNQ was shared by κ-CN in all four different species' milk. Epitope results may provide targets of allergenic fragments for reducing milk allergenicity through physical or/and chemical methods. This study explained the underlying secrets for the high allergenicity of CM to some extent from the perspective of casein and provided new insights for the dairy industry to reduce milk allergy. Furthermore, it provides a new idea and method for comparing the allergenicity of homologous proteins from different species.

SPAD-1, a serine proteinase associated disintegrin from Russell's viper venom disrupts adhesion of MCF7 human breast cancer cells

Serine Proteinase Associated Disintegrin-1 (SPAD-1) is a low molecular mass (26 kDa) positively charged protein purified from Russell's viper venom (RVV) possessing cytotoxic activity on MCF7, human breast cancer cells. Primary sequence analysis of the protein confirms that it is a novel Snake Venom Serine Proteinase (SVSP) and a member of the trypsin family. SPAD-1 contains a conserved triad of Histidine (H), Aspartic acid(D) and Serine(S) residues at its active site for proteinase activity and also an adjacent histidine-glycine-aspartic acid (HGD) disintegrin-like motif. The serine proteinase and disintegrin parts are functionally active and independent. SPAD-1 showed proteolytic digestion of fibrinogen and fibronectin, but laminin digestion was below the detectable limit. Proteolytically inactivated SPAD-1 inhibited collagen and ADP-induced platelet aggregation. This study proposes considering Serine Proteinase Associated Disintegrin (SPAD) as a new group of snake venom proteins. Members of this group contain a serine proteinase catalytic triad and a disintegrin-like motif. SPAD-1 caused visible morphological changes in MCF7 cells, including a reduction of the cell-to-cell attachments, rounding of cell shape and death, in vitro. SPAD-1 also showed a dose-dependent significant decrease in the invasive potency of breast cancer cells. Confocal microscopic analysis revealed the breakage of nuclei of the SPAD-1-treated cells. SPAD-1 also increased cell detachment from the poly L-lysine-coated, laminin-coated and fibronectin-coated culture plate matrices, confirming the disintegrin activity. This study concludes that SPAD-1 may be a good candidate for anti-tumour drug design in the future.

A Shared Anchor on Primary Sigma Factor SigA by the WhiB‐Like Proteins

WhiB‐like (Wbl) proteins are a unique group of iron‐sulfur cluster ([4Fe4S])‐containing transcription factors exclusive to actinobacteria. They play crucial roles in the virulence, survival, and propagation of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. Seven Wbl proteins (WhiB1‐WhiB7) are found in Mtbwith diverse regulatory roles. All Mtb Wbl proteins except for WhiB5 have been shown to interact with the conserved region 4 of the primary sigma factor (SigAr4) in RNA polymerase holoenzyme. Our recent structural and biochemical analyses indicate that two Wbl proteins, WhiB1 and WhiB7, bind to the same site on SigAr4 unexpectedly through tight hydrophobic interactions involving several conserved aromatic residues of the Wbl proteins and His516 of SigAr4. Substitution of these aromatic residues in either WhiB1 or WhiB7 at the molecular interface abolishes their binding to SigAr4. Here, we hypothesize that all Mtb Wbl proteins share a similar molecular interface when in complex with SigAr4, based on the primary sequence analysis and structural modeling. Using the site‐directed mutagenesis and the in vitro protein‐protein interaction assays, we confirm that all Mtb Wbl proteins, including WhiB5, bind to the same site on SigA centered on His516, and the conserved aromatic residues within the Fe‐S cluster binding pocket are required for SigA binding. The results from this study will offer insights into how the Wbl proteins are utilized in Mtb to orchestra gene expression in response to environmental cues in the host.

Taxonomic Revision of the pulcherrima Clade of Metschnikowia (Fungi): Merger of Species

The type strains of 10 small-spored species of the ascomycetous yeast genus Metschnikowia usually form a compact group on the phylogenetic trees inferred from barcode sequences. Based on the name of the species, which was described first (Metschnikowia pulcherrima), the group is frequently referred to as the pulcherrima clade. All strains produce the iron-chelate pigment pulcherrimin and have antagonistic effects on many microorganisms. Recent results of molecular phylogenetic, genetic, and genomic research raised doubts about the taxonomic division of the clade. Those data—combined with results obtained in this study by comprehensive analysis of primary and secondary barcode sequences, physiological tests, and hybridisation experiments—demonstrate that the species cannot be distinguished from each other by the criteria of any of the phenotypic, phylogenetic, and biological species concepts. Therefore, I propose that the species of the pulcherrima clade be merged into one species under the oldest species name, M. pulcherrima.

In silico screening and epitope mapping of leptospiral outer membrane protein—Lsa46

Leptospirosis is one of the neglected diseases caused by the spirochete, Leptospira interrogans. Leptospiral surface adhesion (Lsa) proteins are surface exposed outer membrane proteins present in the pathogen. It acts as laminin and plasminogen binding proteins which enable them to infect host cells. The major target for the development of vaccine in the current era focuses on surface exposed outer membrane proteins, as they can induce strong and fast immune response in hosts. Therefore, the present study mapped the potential epitopes of the Leptospiral outer membrane proteins, mainly the surface adhesion proteins. Protein sequence analysis of Lsa proteins was done by in silico methods. The primary protein sequence analysis revealed Lsa46 as a suitable target which can be a potent Leptospiral vaccine candidate. Its structure was modelled by threading based method in I-TASSER server and validated by Ramachandran plot. The predicted epitope’s interactions with human IgG, IgM(Fab) and T-cell receptor TCR(αβ) were performed by molecular docking studies using Biovia Discovery studio 2018. One of the predicted B-cell epitopes and the IgG showed desirable binding interactions, while four of the predicted B-cell epitopes and T-cell epitopes showed desirable binding interactions with IgM and TCR respectively. The molecular dynamic simulation studies carried out with the molecular docked complexes gave minimized energies indicating stable interactions. The structural analysis of the entire simulated complex showed a stable nature except for one of the Epitope-IgM complex. Further the binding free energy calculation of eight receptor-ligand complex predicted them energetically stable. The results of the study help in elucidating the structural and functional characterization of Lsa46 for epitope-based vaccine design. Communicated by Ramaswamy H. Sarma.

Explicating genetic diversity based on ITS characterization and determination of antioxidant potential in sea buckthorn (Hippophae spp.).

Sea buckthorn (Hippophae) is in the focus of interest mainly for its positive effects on health of both human and animal organisms. Due to the similarities in vegetative morphology, Hippophae species are often misidentified. Therefore, current study was focused on ITS based sequence characterization of sea buckthorn species and comparative biochemical evaluation for its antioxidant properties. DNA was extracted from leaf samples. Primer pairs K-Lab-SeaBukRhm-ITS1F1- K-Lab-SeaBukRhm-ITS1R1 and K-LabSeaBukTib- ITSF1- K-LabSeaBukTib-ITSR1 were used for PCR amplification. The purified PCR products were outsourced for sequencing. Phylogenetic tree was constructed based on neighbor-joining (NJ) method. Moreover, comparison of antioxidant potential of leaves of two sea buckthorn species (Hippophae rhamnoides and Hippophae tibetana) collected from different regions of Ladakh viz., Stakna, Nubra, DRDO Leh and Zanskar was determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2-azino-bis (3- ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS), and Total antioxidant capacity (TAC) by phosphomolybdenum assays. The present investigation led to the differentiation of two sea buckthorn species viz., H. rhamnoides and H. tibetana based on Internal Transcribed Spacer (ITS) region. Moreover, significant variation was observed in antioxidant potential of leaf extracts collected from different regions. Primary ITS sequence analysis was found to be powerful tool for identification and genetic diversity studies in sea buckthorn. Leaves of sea buckthorn have pronounced antioxidant properties and can be used in food, neutraceuticals and pharmaceutical industries etc. The current study will pave the way to discover small bioactive molecules responsible for antioxidant and anticancer properties in sea buckthorn.

Species-Specific Quality Control, Assembly and Contamination Detection in Microbial Isolate Sequences with AQUAMIS.

Sequencing of whole microbial genomes has become a standard procedure for cluster detection, source tracking, outbreak investigation and surveillance of many microorganisms. An increasing number of laboratories are currently in a transition phase from classical methods towards next generation sequencing, generating unprecedented amounts of data. Since the precision of downstream analyses depends significantly on the quality of raw data generated on the sequencing instrument, a comprehensive, meaningful primary quality control is indispensable. Here, we present AQUAMIS, a Snakemake workflow for an extensive quality control and assembly of raw Illumina sequencing data, allowing laboratories to automatize the initial analysis of their microbial whole-genome sequencing data. AQUAMIS performs all steps of primary sequence analysis, consisting of read trimming, read quality control (QC), taxonomic classification, de-novo assembly, reference identification, assembly QC and contamination detection, both on the read and assembly level. The results are visualized in an interactive HTML report including species-specific QC thresholds, allowing non-bioinformaticians to assess the quality of sequencing experiments at a glance. All results are also available as a standard-compliant JSON file, facilitating easy downstream analyses and data exchange. We have applied AQUAMIS to analyze ~13,000 microbial isolates as well as ~1000 in-silico contaminated datasets, proving the workflow’s ability to perform in high throughput routine sequencing environments and reliably predict contaminations. We found that intergenus and intragenus contaminations can be detected most accurately using a combination of different QC metrics available within AQUAMIS.

Purification and Crystallographic Analysis of a Novel Cold-Active Esterase (HaEst1) from Halocynthiibacter arcticus

This report deals with the purification, characterization, and a preliminary crystallographic study of a novel cold-active esterase (HaEst1) from Halocynthiibacter arcticus. Primary sequence analysis reveals that HaEst1 has a catalytic serine in G-x-S-x-G motif. The recombinant HaEst1 was cloned, expressed, and purified. SDS-PAGE and zymographic analysis were carried out to characterize the properties of HaEst1. A single crystal of HaEst1 was obtained in a solution containing 10% (w/v) PEG 8000/8% ethylene glycol, 0.1 M Hepes-NaOH, pH 7.5. Diffraction data were collected to 2.10 Å resolution with P21 space group. The final Rmerge and Rp.i.m values were 7.6% and 3.5% for 50–2.10 Å resolution. The unit cell parameters were a = 35.69 Å, b = 91.21 Å, c = 79.15 Å, and β = 96.9°.

Effects of Conserved Wedge Domain Residues on DNA Binding Activity of Deinococcus radiodurans RecG Helicase.

Deinococcus radiodurans is extremely resistant to ionizing radiation and has an exceptional ability to repair DNA damage caused by various DNA-damaging agents. D. radiodurans uses the same DNA-repair strategies as other prokaryotes, but certain proteins involved in the classical DNA repair machinery have characteristics different from their counterparts. RecG helicase, which unwinds a variety of branched DNA molecules, such as Holliday junctions (HJ) and D-loops, plays important roles in DNA repair, recombination, and replication. Primary sequence analysis of RecG from a number of bacterial species revealed that three amino acids (QPW) in the DNA-binding wedge domain (WD) are well-conserved across the Deinococcus RecG proteins. Interactions involving these conserved residues and DNA substrates were predicted in modeled domain structures of D. radiodurans RecG (DrRecG). Compared to the WD of Escherichia coli RecG protein (EcRecG) containing FSA amino acids corresponding to QPW in DrRecG, the HJ binding activity of DrRecG-WD was higher than that of EcRecG-WD. Reciprocal substitution of FSA and QPW increased and decreased the HJ binding activity of the mutant WDs, EcRecG-WDQPW, and DrRecG-WDFSA, respectively. Following γ-irradiation treatment, the reduced survival rate of DrRecG mutants (ΔrecG) was fully restored by the expression of DrRecG, but not by that of EcRecG. EcRecGQPW also enhanced γ-radioresistance of ΔrecG, whereas DrRecGFSA did not. ΔrecG cells complemented in trans by DrRecG and EcRecGQPW reconstituted an intact genome within 3 h post-irradiation, as did the wild-type strain, but ΔrecG with EcRecG and DrRecGFSA exhibited a delay in assembly of chromosomal fragments induced by γ-irradiation. These results suggested that the QPW residues facilitate the association of DrRecG with DNA junctions, thereby enhancing the DNA repair efficiency of DrRecG.

Structural analysis, virtual screening and molecular simulation to identify potential inhibitors targeting 2'-O-ribose methyltransferase of SARS-CoV-2 coronavirus

SARS-CoV-2, an emerging coronavirus, has spread rapidly around the world, resulting in over ten million cases and more than half a million deaths as of July 1, 2020. Effective treatments and vaccines for SARS-CoV-2 infection do not currently exist. Previous studies demonstrated that nonstructural protein 16 (nsp16) of coronavirus is an S-adenosyl methionine (SAM)-dependent 2’-O-methyltransferase (2’-O-MTase) that has an important role in viral replication and prevents recognition by the host innate immune system. In the present study, we employed structural analysis, virtual screening, and molecular simulation approaches to identify clinically investigated and approved drugs which can act as promising inhibitors against nsp16 2′-O-MTase of SARS-CoV-2. Comparative analysis of primary amino acid sequences and crystal structures of seven human CoVs defined the key residues for nsp16 2-O’-MTase functions. Virtual screening and docking analysis ranked the potential inhibitors of nsp16 from more than 4,500 clinically investigated and approved drugs. Furthermore, molecular dynamics simulations were carried out on eight top candidates, including Hesperidin, Rimegepant, Gs-9667, and Sonedenoson, to calculate various structural parameters and understand the dynamic behavior of the drug-protein complexes. Our studies provided the foundation to further test and repurpose these candidate drugs experimentally and/or clinically for COVID-19 treatment. Communicated by Ramaswamy H. Sarma

Mycobacterium tuberculosis Survival in J774A.1 Cells Is Dependent on MenJ Moonlighting Activity, Not Its Enzymatic Activity.

MenJ, a flavoprotein oxidoreductase, is responsible for the saturation of the β-isoprene unit of mycobacterial menaquinone, resulting in the conversion of menaquinone with nine isoprene units (MK-9) to menaquinone with nine isoprene units where the double bond in the second unit is reduced [MK-9(II-H2)]. The hydrogenation of MK-9 increases the efficiency of the mycobacterial electron transport system, whereas the deletion of MenJ results in decreased survival of the bacteria inside J774A.1 macrophage-like cells but is not required for growth in culture. Thus, it was suggested that MenJ may represent a contextual drug target in M. tuberculosis, that is, a drug target that is valid only in the context of an infected macrophage. However, it was unclear if the conversion of MK-9 to MK-9(II-H2) or the MenJ protein itself was responsible for bacterial survival. In order to resolve this issue, a plasmid expressing folded, full-length, inactive MenJ was engineered. Primary sequence analysis data revealed that MenJ shares conserved FAD binding, NADH binding, and catalytic and C-terminal motifs with archaeal geranylgeranyl reductases. A MenJ mutant deficient in any one of these motifs is devoid of reductase activity. Therefore, point mutations of highly conserved amino acids in the conserved motifs were generated and the recombinant proteins were monitored for conformational changes by circular dichroism and oxidoreductase activity. The mutational analysis indicates that amino acids tryptophan 215 (W215) and cysteine 46 (C46) of M. tuberculosis MenJ, conserved in known archaeal geranylgeranyl reductases and putative menaquinone saturases, are essential to the hydrogenation of MK-9. The mutation of either C46 to serine (C46S) or W215 to leucine (W215L) in MenJ completely abolishes the catalytic activity in vitro, and menJ knockout strains of M. tuberculosis expressing either the C46S or W215L mutant protein are unable to convert MK-9 to MK-9(II-H2) but survive inside the J774A.1 cells. Thus, surprisingly, the survival of M. tuberculosis in J774A.1 cells is dependent on the expression of MenJ rather than its oxidoreductase activity, the conversion of MK-9 to MK-9(II-H2) as previously hypothesized. Overall, the current data suggest that MenJ is a moonlighting protein.

Characterization of the second type of aciniform spidroin (AcSp2) provides new insight into design for spidroin-based biomaterials

Spiders spin a range of silks from different glands for distinct functions, and each silk type exhibits distinct material properties. Silk extruded by the aciniform gland is used for prey wrapping and egg case construction and displays high toughness and extensibility. So far, only the aciniform spidroin 1 (AcSp1) gene which was firstly identified as a silk gene in aciniform gland has been obtained. Here we present the gene sequence for the second type of full-length aciniform silk protein, AcSp2. Analysis of the AcSp2 primary sequence reveals relatively conserved terminal regions and a distinct repetitive sequence relative to AcSp1. A fraction of the gene can be expressed in recombinant systems. Secondary structure analysis of the recombinant AcSp2 protein in solution reveals that the protein adopts mainly an α-helical conformation. Artificial spinning of recombinant AcSp2 demonstrates that the spidroins can be spun into fine fibers which display up to 142% extensibility. The silk fibers are dominated by β-sheet and β-turn secondary structures. Moreover, the mechanical data collected from these synthetic fibers revealed that the mechanical properties are partly correlated with the molecular weights. Overall, our studies enrich our knowledge of spidroin gene family members and provide a new insight into creation of high-performance silk fibers for next generation biomaterials. Statement of SignificanceIn this study, we presented the second type of aciniform silk protein (AcSp2) gene sequence of orb-weaving spider Araneus ventricosus, expanding the spider silk gene family members. The primary structure revealed the central repetitive sequence of the new spidroin gene is distinctly different from other AcSp1 genes. Characterization of the recombinant minispidroin fibers of AcSp2 revealed the mechanical properties are partly correlate with the molecular weights, and the spidroins can be spun into fine fibers which display up to 142% extensibility. Overall, our studies enrich our knowledge of spidroin gene family members and provide a new insight into creation of high-performance silk fibers for next generation biomaterials.

Prioritization of Mur family drug targets against A. baumannii and identification of their homologous proteins through molecular phylogeny, primary sequence, and structural analysis

BackgroundThe World Health Organization (WHO) report stated that Acinetobacter baumannii had been classified as one of the most important pathogenic bacteria causing nosocomial infection in hospital patients due to multi-drug resistance (MDR). It is vital to find out new bacterial drug targets and annotated their structure and function for the exploration of new anti-bacterial agents. The present study utilized a systematic route to prioritize the potential drug targets that belong to Mur family of Acinetobacter baumannii and identify their homologous proteins using a computational approach such as sequence similarity search, multiple sequence alignment, phylogenetic analysis, protein sequence, and protein structure analysis. ResultsFrom the results of protein sequence analysis of eight Mur family proteins, they divided into three main enzymatic classes namely transferases (MurG, MurA and MraY), ligases (MurC, MurD, MurE, and MurF), and oxidoreductase (MurB). Based on the results of intra-comparative protein sequence analysis and enzymatic classification, we have chosen MurB, MurE, and MurG as the prioritized drug targets from A. baumannii and subjected them for further detailed studies of inter-species comparison. This inter-species comparison help us to explore the sequential and structural properties of homologous proteins in other species and hence, opens a gateway for new target identification and using common inhibitor for different bacterial species caused by various diseases. The pairwise sequence alignment results between A. baumannii’s MurB with A. calcoaceticus’s MurB, A. baumannii’s MurE with A. seifertii’s MurE, and A. baumannii’s MurG with A. pittii’s MurG showed that every group of the proteins are highly similar with each other and they showed sequence identity of 95.7% and sequence similarity of 97.2%. ConclusionTogether with the results of secondary and three-dimensional structure predictions explained that three selected proteins (MurB, MurE, and MurG) from A. baumannii and their related proteins (AcMurB, AsMurE, and ApMurG) belong to mixed αβ class and they are very similar.