Halophiles are extremophilic microorganisms that grow optimally at high salt concentrations by producing a myriad of equally halotolerant enzymes. Structural haloadaptation of these enzymes adept to thriving under high-salt environments, though are not fully understood. Herein, the study attempts an in silico investigation to identify and comprehend the evolutionary structural adaptation of a halotolerant dehalogenase, DehHX (GenBank accession number: KR297065) of the halotolerant Pseudomonas halophila, over its non-halotolerant counterpart, DehMX1 (GenBank accession number KY129692) produced by Pseudomonas aeruginosa. GC content of the halotolerant DehHX DNA sequence was distinctively higher (58.9%) than the non-halotolerant dehalogenases (55% average GC). Its acidic residues, Asp and Glu were 8.27% and 12.06%, respectively, compared to an average 5.5% Asp and 7% Glu, in the latter; but lower contents of basic and hydrophobic residues in the DehHX. The secondary structure of DehHX interestingly revealed a lower incidence of α-helix forming regions (29%) and a higher percentage of coils (57%), compared to 49% and 29% in the non-halotolerant homologues, respectively. Simulation models showed the DehHX is stable under a highly saline environment (25% w/v) by adopting a highly negative-charged surface with a concomitant weakly interacting hydrophobic core. The study thus, established that a halotolerant dehalogenase undergoes notable evolutionary structural changes related to GC content over its non-halotolerant counterpart, in order to adapt and thrive under highly saline environments.Communicated by Ramaswamy H. Sarma.
Matched MeSH terms: Protein Conformation, alpha-Helical
A number of living creatures in the Antarctic region have developed characteristic adaptation of cold weather by producing antifreeze proteins (AFP). Antifreeze peptide (Afp1m) fragment have been designed in the sequence of strings from native proteins. The objectives of this study were to assess the properties of Afp1m to cryopreserve skin graft at the temperature of -10 °C and -20 °C and to assess sub-zero injuries in Afp1m cryopreserved skin graft using light microscopic techniques. In the present study, a process was developed to cryopreserve Sprague-Dawley (SD) rat skin grafts with antifreeze peptide, Afp1m, α-helix peptide fragment derived from Glaciozyma antractica yeast. Its viability assessed by different microscopic techniques. This study also described the damages caused by subzero temperatures (-10 and -20 °C) on tissue cryopreserved in different concentrations of Afp1m (0.5, 1, 2, 5 and 10 mg/mL) for 72 h. Histological scores of epidermis, dermis and hypodermis of cryopreserved skin grafts showed highly significant difference (p
Matched MeSH terms: Protein Conformation, alpha-Helical
Macrobrachium rosenbergii nodavirus (MrNV) is the causative agent of white tail disease (WTD) which seriously impedes the production of the giant freshwater prawn and has a major economic impact. MrNV contains two segmented RNA molecules, which encode the RNA dependent RNA polymerase (RdRp) and the capsid protein (MrNV-CP) containing 371 amino acid residues. MrNV-CP comprises of the Shell (S) and the Protruding (P) domains, ranging from amino acid residues 1-252 and 253-371, respectively. The P-domain assembles into dimeric protruding spikes, and it is believed to be involved in host cell attachment and internalization. In this study, the recombinant P-domain of MrNV-CP was successfully cloned and expressed in Escherichia coli, purified with an immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC) up to ~90% purity. Characterization of the purified recombinant P-domain with SEC revealed that it formed dimers, and dynamic light scattering (DLS) analysis demonstrated that the hydrodynamic diameter of the dimers was ~6 nm. Circular dichroism (CD) analysis showed that the P-domain contained 67.9% of beta-sheets, but without alpha-helical structures. This is in good agreement with the cryo-electron microscopic analysis of MrNV which demonstrated that the P-domain contains only beta-stranded structures. Our findings of this study provide essential information for the production of the P-domain of MrNV-CP that will aid future studies particularly studies that will shed light on anti-viral drug discovery and provide an understanding of virus-host interactions and the viral pathogenicity.
Matched MeSH terms: Protein Conformation, alpha-Helical
Fatty acid desaturase catalyzes the desaturation reactions by insertion of double bonds into the fatty acyl chain, producing unsaturated fatty acids. Though soluble fatty acid desaturases have been studied widely in advanced organisms, there are very limited studies of membrane fatty acid desaturases due to the difficulty of generating recombinant desaturase. Brassica napus is a rapeseed, which possesses a range of different membrane-bound desaturases capable of producing fatty acids including Δ3, Δ4, Δ8, Δ9, Δ12, and Δ15 fatty acids. The 1155 bp open reading frame of Δ12 fatty acid desaturase (FAD12) from Brassica napus codes for 383 amino acid residues with a molecular weight of 44 kDa. It was expressed in Escherichia coli at 37 °C in soluble and insoluble forms when induced with 0.5 mM IPTG. Soluble FAD12 has been purified using Ni2+-Sepharose affinity chromatography with a total protein yield of 0.728 mg/mL. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that desaturase activity of FAD12 could produce linoleic acid from oleic acid at a retention time of 17.6 with a conversion rate of 47%. Characterization of purified FAD12 revealed the optimal temperature of FAD12 was 50 °C with 2 mM preferred substrate concentration of oleic acid. Analysis of circular dichroism (CD) showed FAD12 was made up of 47.3% and 0.9% of alpha-helix and β-sheet secondary structures. The predicted Tm value was 50.2 °C.
Matched MeSH terms: Protein Conformation, alpha-Helical
Antifreeze proteins (AFPs) are specific proteins, glycopeptides, and peptides made by different organisms to allow cells to survive in sub-zero conditions. AFPs function by reducing the water's freezing point and avoiding ice crystals' growth in the frozen stage. Their capability in modifying ice growth leads to the stabilization of ice crystals within a given temperature range and the inhibition of ice recrystallization that decreases the drip loss during thawing. This review presents the potential applications of AFPs from different sources and types. AFPs can be found in diverse sources such as fish, yeast, plants, bacteria, and insects. Various sources reveal different α-helices and β-sheets structures. Recently, analysis of AFPs has been conducted through bioinformatics tools to analyze their functions within proper time. AFPs can be used widely in various aspects of application and have significant industrial functions, encompassing the enhancement of foods' freezing and liquefying properties, protection of frost plants, enhancement of ice cream's texture, cryosurgery, and cryopreservation of cells and tissues. In conclusion, these applications and physical properties of AFPs can be further explored to meet other industrial players. Designing the peptide-based AFP can also be done to subsequently improve its function.
Matched MeSH terms: Protein Conformation, alpha-Helical
Schistosomes are parasitic blood flukes that infect approximately 250 million people worldwide. The disease known as schistosomiasis, is the second most significant tropical parasitic disease after malaria. Praziquantel is the only effective drug currently licensed for schistosomiasis and there are concerns about resistance to the drug. There has been much effort to develop vaccines against schistosomiasis to produce long-term protection in endemic regions. Surface-associated proteins, and in particular, those expressed in the body wall, or tegument, have been proposed as potential vaccine targets. Of these, annexins are thought to be of integral importance for the stability of this apical membrane system. Here, we present the structural and immunobiochemical characterization of four homologous annexins namely annexin B30, annexin B5a, annexin B7a and annexin B5b from S. mansoni. Bioinformatics analysis showed that there was no signal peptide predicted for any annexin in this study. Further analysis showed that each of all four annexin protein possesses a primary structure consisting of a short but variable N-terminal region and a long C-terminal core containing four homologous annexin repeats (I-IV), which contain five alpha-helices. The life cycle expression profile of each annexin was assessed using quantitative PCR. The results showed that the overall transcript levels of the each of four homologous annexins were relatively low in the egg stage, but increased gradually after the transition of cercariae (the invasive schistosome larvae) to schistosomula (the post-invasive larvae). Circular dichroism (CD) demonstrated that rAnnexin B30, rAnnexin B5a and rAnnexin 7a were folded, showing a secondary structure content rich in alpha-helices. The membrane binding affinity was enhanced when rAnnexin B30, rAnnexin B5a and rAnnexin 7a was incubated in the presence of Ca2+. All annexin members evaluated in this study were immunolocalized to the tegument, with immunoreactivity also occurring in cells and in muscle of adult parasites. All four recombinant annexins were immunoreactive and they were recognized by the sera of mice infected with S. mansoni. In conclusion, the overall results present the molecular characterization of annexin B30, annexin B5a, annexin B7a and annexin B5b from S. mansoni in host-parasite interactions and strongly suggest that the molecules could be useful candidates for vaccine or diagnostic development.
Matched MeSH terms: Protein Conformation, alpha-Helical
Dehalogenases are of high interest due to their potential applications in bioremediation and in synthesis of various industrial products. DehL is an L-2-haloacid dehalogenase (EC 3.8.1.2) that catalyses the cleavage of halide ion from L-2-halocarboxylic acid to produce D-2-hydroxycarboxylic acid. Although DehL utilises the same substrates as the other L-2-haloacid dehalogenases, its deduced amino acid sequence is substantially different (<25%) from those of the rest L-2-haloacid dehalogenases. To date, the 3D structure of DehL is not available. This limits the detailed understanding of the enzyme's reaction mechanism. The present work predicted the first homology-based model of DehL and defined its active site. The monomeric unit of the DehL constitutes α/β structure that is organised into two distinct structural domains: main and subdomains. Despite the sequence disparity between the DehL and other L-2-haloacid dehalogenases, its structural model share similar fold as the experimentally solved L-DEX and DehlB structures. The findings of the present work will play a crucial role in elucidating the molecular details of the DehL functional mechanism.
Matched MeSH terms: Protein Conformation, alpha-Helical
Crystal structures of holo vitamin D receptor (VDR) revealed a canonical conformation in which the ligand is entrapped in a hydrophobic cavity buried in the ligand-binding domain (LBD). The mousetrap model postulates that helix 12 is positioned away from the domain to expose the interior cavity. However, the extended form of helix 12 is likely due to artifacts during crystallization. In this study, we set out to investigate conformational dynamics of apo VDR using molecular dynamics simulation on microsecond timescale. Here we show the neighboring backbones of helix 2-helix 3n and beta strand 2-helix 6 of LBD, instead of the helix 12, undergo large-scale motion, possibly gating the entrance of ligand to the ligand binding domain. Docking analysis to the simulated open structure of VDR with the estimated free energy of -37.0 kJ/mol, would emphasise the role of H2-H3n and S2-H6 in facilitating the entrance of calcitriol to the LBD of VDR.
Matched MeSH terms: Protein Conformation, alpha-Helical
The IntFOLD server provides a unified resource for the automated prediction of: protein tertiary structures with built-in estimates of model accuracy (EMA), protein structural domain boundaries, natively unstructured or disordered regions in proteins, and protein-ligand interactions. The component methods have been independently evaluated via the successive blind CASP experiments and the continual CAMEO benchmarking project. The IntFOLD server has established its ranking as one of the best performing publicly available servers, based on independent official evaluation metrics. Here, we describe significant updates to the server back end, where we have focused on performance improvements in tertiary structure predictions, in terms of global 3D model quality and accuracy self-estimates (ASE), which we achieve using our newly improved ModFOLD7_rank algorithm. We also report on various upgrades to the front end including: a streamlined submission process, enhanced visualization of models, new confidence scores for ranking, and links for accessing all annotated model data. Furthermore, we now include an option for users to submit selected models for further refinement via convenient push buttons. The IntFOLD server is freely available at: http://www.reading.ac.uk/bioinf/IntFOLD/.
Matched MeSH terms: Protein Conformation, alpha-Helical
In infections by apicomplexan parasites including Plasmodium, Toxoplasma gondii, and Eimeria, host interactions are mediated by proteins including families of membrane-anchored cysteine-rich surface antigens (SAGs) and SAG-related sequences (SRS). Eimeria tenella causes caecal coccidiosis in chickens and has a SAG family with over 80 members making up 1% of the proteome. We have solved the structure of a representative E. tenella SAG, EtSAG19, revealing that, despite a low level of sequence similarity, the entire Eimeria SAG family is unified by its three-layer αβα fold which is related to that of the CAP superfamily. Furthermore, sequence comparisons show that the Eimeria SAG fold is conserved in surface antigens of the human coccidial parasite Cyclospora cayetanensis but this fold is unrelated to that of the SAGs/SRS proteins expressed in other apicomplexans including Plasmodium species and the cyst-forming coccidia Toxoplasma gondii, Neospora caninum and Besnoitia besnoiti. However, despite having very different structures, Consurf analysis showed that Eimeria SAG and Toxoplasma SRS families each exhibit marked hotspots of sequence hypervariability that map to their surfaces distal to the membrane anchor. This suggests that the primary and convergent purpose of the different structures is to provide a platform onto which sequence variability can be imposed.
Matched MeSH terms: Protein Conformation, alpha-Helical
Extracellular signal-regulated kinases 1 and 2 (ERK1/2) play key roles in promoting cell survival and proliferation through the phosphorylation of various substrates. Remarkable antitumour activity is found in many inhibitors that act upstream of the ERK pathway. However, drug-resistant tumour cells invariably emerge after their use due to the reactivation of ERK1/2 signalling. ERK1/2 inhibitors have shown clinical efficacy as a therapeutic strategy for the treatment of tumours with mitogen-activated protein kinase (MAPK) upstream target mutations. These inhibitors may be used as a possible strategy to overcome acquired resistance to MAPK inhibitors. Here, we report a class of repeat proteins-designed ankyrin repeat protein (DARPin) macromolecules targeting ERK2 as inhibitors. The structural basis of ERK2-DARPin interactions based on molecular dynamics (MD) simulations was studied. The information was then used to predict stabilizing mutations employing a web-based algorithm, MAESTRO. To evaluate whether these design strategies were successfully deployed, we performed all-atom, explicit-solvent molecular dynamics (MD) simulations. Two mutations, Ala → Asp and Ser → Leu, were found to perform better than the original sequence (DARPin E40) based on the associated energy and key residues involved in protein-protein interaction. MD simulations and analysis of the data obtained on these mutations supported our predictions.
Matched MeSH terms: Protein Conformation, alpha-Helical
TylP is one of five regulatory proteins involved in the regulation of antibiotic (tylosin) production, morphological and physiological differentiation in Streptomyces fradiae. Its function is similar to those of various γ-butyrolactone receptor proteins. In this report, N-terminally His-tagged recombinant TylP protein (rTylP) was overproduced in Escherichia coli and purified to homogeneity. The rTylP protein was crystallized from a reservoir solution comprising 34%(v/v) ethylene glycol and 5%(v/v) glycerol. The protein crystals diffracted X-rays to 3.05 Å resolution and belonged to the trigonal space group P3121, with unit-cell parameters a = b = 126.62, c = 95.63 Å.
Matched MeSH terms: Protein Conformation, alpha-Helical
A common drug repositioning strategy is the re-application of an existing drug to address alternative targets. A crucial aspect to enable such repurposing is that the drug's binding site on the original target is similar to that on the alternative target. Based on the assumption that proteins with similar binding sites may bind to similar drugs, the 3D substructure similarity data can be used to identify similar sites in other proteins that are not known targets. The Drug ReposER (DRug REPOSitioning Exploration Resource) web server is designed to identify potential targets for drug repurposing based on sub-structural similarity to the binding interfaces of known drug binding sites. The application has pre-computed amino acid arrangements from protein structures in the Protein Data Bank that are similar to the 3D arrangements of known drug binding sites thus allowing users to explore them as alternative targets. Users can annotate new structures for sites that are similarly arranged to the residues found in known drug binding interfaces. The search results are presented as mappings of matched sidechain superpositions. The results of the searches can be visualized using an integrated NGL viewer. The Drug ReposER server has no access restrictions and is available at http://mfrlab.org/drugreposer/.
Matched MeSH terms: Protein Conformation, alpha-Helical
MTH1 is an enzyme that hydrolyzes 8-oxo-dGTP, which is an oxidatively damaged nucleobase, into 8-oxo-dGMP in nucleotide pools to prevent its mis-incorporation into genomic DNA. Selective and potent MTH1-binding molecules have potential as biological tools and drug candidates. We recently developed 8-halogenated 7-deaza-dGTP as an 8-oxo-dGTP mimic and found that it was not hydrolyzed, but inhibited enzyme activity. To further increase MTH1 binding, we herein designed and synthesized 7,8-dihalogenated 7-deaza-dG derivatives. We successfully synthesized multiple derivatives, including substituted nucleosides and nucleotides, using 7-deaza-dG as a starting material. Evaluations of the inhibition of MTH1 activity revealed the strong inhibitory effects on enzyme activity of the 7,8-dihalogenated 7-deaza-dG derivatives, particularly 7,8-dibromo 7-daza-dGTP. Based on the results obtained on kinetic parameters and from computational docking simulating studies, these nucleotide analogs interacted with the active site of MTH1 and competitively inhibited the substrate 8-oxodGTP. Therefore, novel properties of repair enzymes in cells may be elucidated using new compounds.
Matched MeSH terms: Protein Conformation, alpha-Helical
Fuculose-1-phosphate aldolase (FucA) catalyses the reversible cleavage of L-fuculose 1-phosphate to dihydroxyacetone phosphate (DHAP) and L-lactaldehyde. This enzyme from mesophiles and thermophiles has been extensively studied; however, there is no report on this enzyme from a psychrophile. In this study, the gene encoding FucA from Glaciozyma antarctica PI12 (GaFucA) was cloned and the enzyme was overexpressed in Escherichia coli, purified and crystallized. The tetrameric structure of GaFucA was determined to 1.34 Å resolution. The overall architecture of GaFucA and its catalytically essential histidine triad are highly conserved among other fuculose aldolases. Comparisons of structural features between GaFucA and its mesophilic and thermophilic homologues revealed that the enzyme has typical psychrophilic attributes, indicated by the presence of a high number of nonpolar residues at the surface and a lower number of arginine residues.
Matched MeSH terms: Protein Conformation, alpha-Helical
Annona muricata Linn or usually identified as soursop is a potential anticancer plant that has been widely reported to contain valuable chemopreventive agents known as annonaceous acetogenins. The antiproliferative and anticancer activities of this tropical and subtropical plant have been demonstrated in cell culture and animal studies. A. muricata L. exerts inhibition against numerous types of cancer cells, involving multiple mechanism of actions such as apoptosis, a programmed cell death that are mainly regulated by Bcl-2 family of proteins. Nonetheless, the binding mode and the molecular interactions of the plant's bioactive constituents have not yet been unveiled for most of these mechanisms. In the current study, we aim to elucidate the binding interaction of ten bioactive phytochemicals of A. muricata L. to three Bcl-2 family of antiapoptotic proteins viz. Bcl-2, Bcl-w and Mcl-1 using an in silico molecular docking analysis software, Autodock 4.2. The stability of the complex with highest affinity was evaluated using MD simulation. We compared the docking analysis of these substances with pre-clinical Bcl-2 inhibitor namely obatoclax. The study identified the potential chemopreventive agent among the bioactive compounds. We also characterized the important interacting residues of protein targets which involve in the binding interaction. Results displayed that anonaine, a benzylisoquinoline alkaloid, showed a high affinity towards the Bcl-2, thus indicating that this compound is a potent inhibitor of the Bcl-2 antiapoptotic family of proteins.
Matched MeSH terms: Protein Conformation, alpha-Helical