Amino Acids Of Subunit Research Articles

Protein phosphorylation and oxidative protein modification promote plant photosystem II disassembly for repair

The light-driven water-splitting reaction of photosystem II exposes its key reaction center core protein subunits to irreversible oxidative photodamage. A rapid repair cycle replaces the photodamaged core subunits in plants, but how the large antenna-core supercomplex structures of plant photosystem II disassemble for repair is not currently understood. We find a specific involvement of phosphorylation in removing the peripheral antenna from the core and monomerization of the dimeric cores. However, monomeric cores disassembled further into smaller subcomplexes even in the absence of phosphorylation as suggestive of other unknown mechanisms of disassembly. Here we show that the oxidative modifications of amino acids in core protein subunits of photosystem II are active mediators of the monomeric core disassembly. Oxidative modifications thus likely disassemble only the damaged monomeric cores, ensuring an economical photosystem disassembly process. Taken together, our results suggest phosphorylation and oxidative modification play distinct roles in photosystem II disassembly and repair.

Cryo-EM structures of type IV pili complexed with nanobodies reveal immune escape mechanisms

Type IV pili (T4P) are prevalent, polymeric surface structures in pathogenic bacteria, making them ideal targets for effective vaccines. However, bacteria have evolved efficient strategies to evade type IV pili-directed antibody responses. Neisseria meningitidis are prototypical type IV pili-expressing Gram-negative bacteria responsible for life threatening sepsis and meningitis. This species has evolved several genetic strategies to modify the surface of its type IV pili, changing pilin subunit amino acid sequence, nature of glycosylation and phosphoforms, but how these modifications affect antibody binding at the structural level is still unknown. Here, to explore this question, we determine cryo-electron microscopy (cryo-EM) structures of pili of different sequence types with sufficiently high resolution to visualize posttranslational modifications. We then generate nanobodies directed against type IV pili which alter pilus function in vitro and in vivo. Cryo-EM in combination with molecular dynamics simulation of the nanobody-pilus complexes reveals how the different types of pili surface modifications alter nanobody binding. Our findings shed light on the impressive complementarity between the different strategies used by bacteria to avoid antibody binding. Importantly, we also show that structural information can be used to make informed modifications in nanobodies as countermeasures to these immune evasion mechanisms.

Effects of simultaneous water-enzyme separation on the physical and chemical properties of peanut oil and protein

Peanut (Arachis hypogaea L.) is widely cultivated as a valuable industrial oilseed. This study aimed to investigate the oil and protein that was extracted from peanuts by aqueous enzymatic method (AEM). Compared with freeze-thaw demulsification (FTD), n-hexane extraction (NHE) and protease demulsification (PD), the fatty acid composition of oil by combination of oleic and citric acids demulsification (COCAD) was similar. The acid value of the oil extracted by new AEM and various demulsification methods was between 0.12 and 0.59 mg KOH/g, the peroxide value was between 0.06 and 0.19 g/100 g, the free fatty acid content was between 0.32% and 0.71%, and the oxidation induction time was between 0.13 and 3.21 h. The content of tocopherol in oil: NHE>FTD>PD>COCAD. The amino acid and protein subunit compositions of peanut protein extracted by AEM (AEMPP), peanut protein isolate (PPI) and commercially available peanut protein (CAPP) were similar. Scanning electron microscope revealed that protein powders morphology of various extraction methods. Compared to CAPP and PPI, β-turns and random coil structure of AEMPP was increased. The solubility, water and oil holding capacity, emulsifying activity index, emulsification stability, foaming property and foam stability of protein: AEMPP > PPI > CAPP. This study provides a theoretical basis for the large-scale industrial application of AEM.

Molecular structures reveal the origin of spectral variation in cryptophyte light harvesting antenna proteins.

In addition to their membrane-bound chlorophyll a/c light-harvesting antenna, the cryptophyte algae have evolved a unique phycobiliprotein antenna system located in the thylakoid lumen. The basic unit of this antenna consists of two copies of an αβ protomer where the α and β subunits scaffold different combinations of a limited number of linear tetrapyrrole chromophores. While the β subunit is highly conserved, encoded by a single plastid gene, the nuclear-encoded α subunits have evolved diversified multigene families. It is still unclear how this sequence diversity results in the spectral diversity of the mature proteins. By careful examination of three newly determined crystal structures in comparison with three previously obtained, we show how the α subunit amino acid sequences control chromophore conformations and hence spectral properties even when the chromophores are identical. Previously we have shown that α subunits control the quaternary structure of the mature αβ.αβ complex (either open or closed), however, each species appeared to only harbor a single quaternary form. Here we show that species of the Hemiselmis genus contain expressed α subunit genes that encode both distinct quaternary structures. Finally, we have discovered a common single-copy gene (expressed into protein) consisting of tandem copies of a small α subunit that could potentially scaffold pairs of light harvesting units. Together, our results show how the diversity of the multigene α subunit family produces a range of mature cryptophyte antenna proteins with differing spectral properties, and the potential for minor forms that could contribute to acclimation to varying light regimes.

The trajectory in catalytic evolution of Rubisco in Posidonia seagrass species differs from terrestrial plants.

The CO2-fixing enzyme Ribulose bisphosphate carboxylase-oxygenase (Rubisco) links the inorganic and organic phases of the global carbon cycle. In aquatic systems, the catalytic adaptation of algae Rubiscos has been more expansive and followed an evolutionary pathway that appears distinct to terrestrial plant Rubisco. Here, we extend this survey to differing seagrass species of the genus Posidonia to reveal how their disjunctive geographical distribution and diverged phylogeny, along with their CO2 concentrating mechanisms (CCMs) effectiveness, have impacted their Rubisco kinetic properties. The Rubisco from Posidonia species showed lower carboxylation efficiencies and lower sensitivity to O2 inhibition than those measured for terrestrial C3 and C4-plant Rubiscos. Compared with the Australian Posidonia species, Rubisco from the Mediterranean Posidonia oceanica had 1.5-2-fold lower carboxylation and oxygenation efficiencies, coinciding with effective CCMs and five Rubisco large subunit amino acid substitutions. Among the Australian Posidonia species, CCM effectiveness was higher in Posidonia sinuosa and lower in the deep-living Posidonia angustifolia, likely related to the 20%-35% lower Rubisco carboxylation efficiency in P. sinuosa and the two-fold higher Rubisco content in P. angustifolia. Our results suggest that the catalytic evolution of Posidonia Rubisco has been impacted by the low CO2 availability and gas exchange properties of marine environments, but with contrasting Rubisco kinetics according to the time of diversification among the species. As a result, the relationships between maximum carboxylation rate and CO2- and O2-affinities of Posidonia Rubiscos follow an alternative path to that characteristic of terrestrial angiosperm Rubiscos.

Isolation, Characterization, and Genome Analysis of a Novel Bacteriophage, Escherichia Phage vB_EcoM-4HA13, Representing a New Phage Genus in the Novel Phage Family Chaseviridae.

Shiga toxin-producing Escherichia coli (STEC) is one of the leading causes of foodborne illnesses in North America and can lead to severe symptoms, with increased fatality risk for young children. While E. coli O157:H7 remains the dominant STEC serotype associated with foodborne outbreaks, there has been an increasing number of non-O157 STEC outbreaks in recent years. For the food industry, lytic bacteriophages offer an organic, self-limiting alternative to pathogen reduction-one that could replace or reduce the use of chemical and physical food processing methods. From EHEC-enriched sewage, we isolated a novel bacteriophage, vB_EcoM-4HA13 (4HA13). Phenotypic characterizations revealed 4HA13 to possess a myoviral morphotype, with a high specificity to non-motile O111 serotype, and a long latent period (90 min). Through genomic analyses, this 52,401-bp dsDNA phage was found to contain 81 CDS, but no detectable presence of antibiotic resistance, integrase, or virulence genes. A BLASTn search for each of the identified 81 CDS yielded homologues with low levels of similarity. Comparison of RNA polymerase and terminase large subunit amino acid sequences led to the proposal and acceptance of a new bacteriophage family, Chaseviridae, with 4HA13 representing a new species and genus. The discovery of this phage has broadened our current knowledge of bacteriophage diversity.

Tailoring protein intrinsic charge by enzymatic deamidation for solubilizing chicken breast myofibrillar protein in water

Inspired by the salt-in effect, the potential use of protein-glutaminase (PG) to increase the intrinsic charges of chicken breast myofibrillar proteins (CMPs) for enhanced water solubility was tested. The degree of deamidation (DD) and solubility of CMPs increased with PG reaction time. Over 60% of CMPs were soluble in water under a DD of 6.5% due to specific conversion of glutamine to glutamic acid. PG deamidation could remarkably increase the net charge of CMPs with a merit in maintaining most of the amino acid and protein subunit compositions. Such a high electrostatic repulsion exerted a transformation of β-sheet into α-helix, unfolded the structure to expose hydrophobic residues, and allowed the dissociation of myofibril and release of subunits (myosin, actin or their oligomers), leading to a stable colloidal state. This work may foster the engineering advances of protein micro-modification in the tailor manufacture of muscle protein-based beverages.

Progesterone activates BK channels by binding to transmembrane amino acids of the channel β1 regulatory subunit

Progesterone (PROG) is a naturally occurring steroid hormone. Emerging data indicate that PROG has non-reproductive functions including roles in neurogenesis, neuro-regeneration and cerebrovascular dilation, the latter being a rationale for PROG use in recovery from traumatic brain injury. PROG actions have been traditionally linked to activation of cytosolic and membrane-bound steroid receptors. However, the molecular mechanisms underlying the vasoactive properties of PROG remain largely unknown.

Rationale Design, Synthesis, and Pharmacological Evaluation of Isatin Analogues as Antiseizure Agents

Background: Epilepsy is one of the most common and devastating neurological diseases affecting about 1% of the world’s population at any time. Herein, we have reported rationale design and synthesis of 5-acetyl-3-((4-substitutedphenyl)imino)indolin-2-one analogues 3(a-k) as antiseizure agents. Objective: These analogues were designed as four component pharmacophoric model by clubbing structural fragments of potent antiepileptic drugs. The Aim of this study is to synthesize structurally designed isatin analogues and screen them for anticonvulsant activity and neurotoxicity. Methods: The compound 3d have exhibited good anticonvulsant activity in preclinical seizure models with better toxicity profile when compared to standard drugs (3d: ED50 = 31.5 mg/kg, MES test; ED50 = 37.4 mg/kg, scPTZ test; TD50 = 384.3 mg/kg,). Compound 3d have also shown good binding affinity at crucial amino acids of GluN1 subunit of NMDAR (Docking score = -9.30) and fit adequately in the cavity of the receptor (Amino acids involved and H-bonding distance = GLY93: acetyl >C=O, 2.38Å; PHE92: acetyl >C=O, 2.22Å; and THR26: >C=O of isatin, 1.71Å). Results of molecular docking supported the structural features present over isatins to persuade potent antiseizure activity. Results: The compound 3d have exhibited good anticonvulsant activity in preclinical seizure models with better toxicity profile when compared to standard drugs (3d: ED50 = 31.5 mg/kg, MES test; ED50 = 37.4 mg/kg, scPTZ test; TD50 = 384.3 mg/kg,). Compound 3d have also shown good binding affinity at crucial amino acids of GluN1 subunit of NMDAR (Docking score = -9.30) and fit adequately in the cavity of the receptor (Amino acids involved and H-bonding distance = GLY93: acetyl >C=O, 2.38Å; PHE92: acetyl >C=O, 2.22Å; and THR26: >C=O of isatin, 1.71Å). Results of molecular docking supported the structural features present over isatins to persuade potent antiseizure activity. Conclusion: Rationale designing strategy, in-vivo pharmacological profile, and computational studies make us anticipate the emergence of these molecules as novel antiseizure agents, which can be further explored to develop probes for the treatment of epilepsy.

Self-Regulation of Cerebral Metabolism and Its Neuroprotective Effect After Hypoxic-Ischemic Injury: Evidence From 1H-MRS.

1H-MRS technology can be used to non-invasively detect the content of cerebral metabolites, to assess the severity of hypoxic-ischemic (HI) injury, and to predict the recovery of compromised neurological function. However, changes to the cerebral self-regulation process after HI are still unclear. This study investigated the changes in cerebral metabolites and the potential relationship with the number of neurons and neural stem/progenitor cells (NSPC) using 1H-MRS, and finally clarifies the self-regulation of cerebral metabolism and neuroprotection after HI injury. Newborn Yorkshire pigs (28 males, 1.0–1.5 kg) aged 3–5 days were used for the HI model in this study. The pigs were randomly divided into the HI group (n = 24) and the control group (n = 4), then the experimental group was subdivided according to different recovery time after HI into the following groups: 0–2 h (n = 4), 2–6 h (n = 4), 6–12 h (n = 4), 12–24 h (n = 4), 24–48 h (n = 4), and 48–72 h (n = 4). Following the HI timepoints, 1H-MRS scans were performed and processed using LCModel software, and brain tissue was immunohistochemically stained for Nestin and NeuN. Immunofluorescence staining of creatine phosphokinase-BB (CK-BB), N-acetylaspartylglutamate synthetase (NAAGS), glutamate carboxypeptidase II (GCP-II), glutamate-cysteine ligase catalytic subunit (GCLC), glutathione synthase (GS), and excitatory amino acid carrier 1 (EAAC1) was then performed. The 1H-MRS results showed that cerebral N-acetylaspartylglutamate (NAAG), glutathione (GSH), and creatine (Cr) content reached their peaks at 12–24 h, which was consistent with the recovery time of hippocampal NSPCs and neurons, indicating a potential neuroprotective effect of NAAG, GSH, and Cr after HI injury.

The transcriptional activator of the bfp operon in EPEC (PerA) interacts with the RNA polymerase alpha subunit

Enteropathogenic E. coli virulence genes are under the control of various regulators, one of which is PerA, an AraC/XylS-like regulator. PerA directly promotes its own expression and that of the bfp operon encoding the genes involved in the biogenesis of the bundle-forming pilus (BFP); it also activates PerC expression, which in turn stimulates locus of enterocyte effacement (LEE) activation through the LEE-encoded regulator Ler. Monomeric PerA directly binds to the per and bfp regulatory regions; however, it is not known whether interactions between PerA and the RNA polymerase (RNAP) are needed to activate gene transcription as has been observed for other AraC-like regulators. Results showed that PerA interacts with the alpha subunit of the RNAP polymerase and that it is necessary for the genetic and phenotypic expression of bfpA. Furthermore, an in silico analysis shows that PerA might be interacting with specific alpha subunit amino acids residues highlighting the direction of future experiments.

AteqTERT expression and specific tissue activity in a 2-year-old complete plant in Agave tequilana in field conditions

Telomerase is a specialized enzyme that attributes to maintaining and lengthening of telomeric length as its primary function. This ribonucleoprotein complex requires two minimum subunits for its in vivo function: the TERT amino acid subunit and TER its ribonucleic subunit. Crystallographic studies have shown that stable interaction between subunits and their domains is essential during the active phase. Plants and humans have shown to be tissue specific and dependent on cell proliferation. In Arabidopsis thaliana (L.) Heynh, the expression of AtTERT was determined to be necessary for activity. The objective of this work is to perform the in silico characterization of TERT in Agave tequilana F.A.C. Weber and evaluate the expression of TERT and the specific tissue activity in 2-year-old plants. To this end, we use the transcriptome library of A. tequilana (Avila de Dios unpublished data) in TPM and qTRAP. We observe in this work that the consensus sequence shows all the specific domains of the TERT subunit. Additionally, we carried out the in silico prediction of the structural model of AteqTERT. The model presents the characteristic TERT ring of the crystallized models in active state of telomerase, biological annotations were observed, such as heavy metals Mg2+, NUC and CH1, and finally we note that, there is no correlation between TERT expression in TPM and telomerase activity in 2-year meristematic tissue.

Identification of novel interacting regions involving calcineurin and nuclear factor of activated T cells

Nuclear factor of activated T cells (NFAT) leads to the transcription of diverse inducible genes involved in many biological processes; therefore, aberrant NFAT expression is responsible for the development and exacerbation of various disorders. Since five isoforms of NFAT (NFATc1-c4, NFAT5) exhibit distinct and overlapping functions, selective control of a part, but not all, of NFAT family members is desirable. By comparing the binding activity of each NFATc1-c4 with its regulatory enzyme, calcineurin (CN), using a quantitative immunoprecipitation assay, we found a new CN-binding region (CNBR) selectively functioning in NFATc1 and NFATc4. This region, termed CNBR3, is located between two preexisting CNBR1 and CNBR2, within the Ca2+ regulatory domain. The nuclear translocation of NFATc1 but not NFATc2 in T cells was suppressed by ectopic expression of CNBR3 and, accordingly, NFATc1-dependent cytokine expression was downregulated. Through competition assays using NFATc1-derived partial peptides and mass spectrometry with photoaffinity technology, we identified 18 amino acids in NFATc1 (Arg258 to Pro275 ) and 13 amino acids in CN catalytic subunit (CNA) (Asn77 to Gly89 ) responsible for CNA/CNBR3 binding in which Cys263 and Asp82 , respectively, played crucial roles. The possible selective regulation of NFAT-mediated biological processes by targeting this new CN/NFAT-binding region is suggested.

Qualitative and Quantitative Determination of MERS-CoV S1-Specific Antibodies Using ELISA.

Indirect enzyme-linked immunosorbent assay (ELISA) enables detection and quantification of antigen-specific antibodies in biological samples such as human or animal sera. Most current MERS-CoV serological assays such as neutralization, immunofluorescence, or protein microarray rely on handling of live MERS-CoV in high containment laboratories, highly trained personnel as well as the need for expensive and special equipment and reagents representing a hurdle for most laboratories especially when resources are limited. In this chapter, we describe a validated and optimized indirect ELISA protocol based on recombinant S1 subunit (amino acids 1–725) of MERS-CoV for qualitative and quantitative determination of MERS-CoV-binding antibodies.

Involvement of sulfur assimilation in the low β subunit content of soybean seed storage protein revealed by comparative transcriptome analysis

The β subunit of soybean [Glycine max (L.) Merr.] seed storage protein is of great significance in sulfur-containing amino acid balance and soybean processing properties. The objective of this study was to elucidate the relationship between the β subunit and sulfur-containing amino acid composition, and the potential regulatory mechanism. The β subunit was independently accumulated in comparison with other major subunits (α/α′, acidic, basic, and A3) during seed filling, and a low level of β subunit content (BSC) was formed during the accumulation process. In low-BSC mature seeds, crude protein, oil content, and fatty acid composition were not changed, but sulfur-containing amino acids (Cys + Met) in the low-BSC seeds increased significantly (by 31.5%), suggesting that an internal regulatory mechanism within seed might be responsible for the rebalance of seed protein composition and that sulfur assimilation might be deeply involved in β subunit accumulation. Transcriptomic analysis revealed that genes involved in anabolism of cysteine, methionine, and glutathione were up-regulated but those involved in the catabolism of these compounds were down-regulated, suggesting a relationship between the elevation of methionine and glutathione and low BSC. Our study sheds light on seed composition in low BSC lines and on the potential molecular regulatory mechanism of β subunit accumulation, broadening our understanding of soybean seed protein synthesis and its regulation.

Development and validation of different indirect ELISAs for MERS-CoV serological testing

Since 2012, MERS-CoV has caused up to 2220 cases and 790 deaths in 27 countries with Saudi Arabia being the most affected country with ~83.1% of the cases and ~38.8% local death rate. Current serological assays such as microneutralization (MN), plaque reduction neutralization, immunofluorescence, protein microarray or pseudoparticle neutralization assays rely on handling of live MERS-CoV in high containment laboratories or need for expensive and special equipment and reagents and highly trained personnel which represent a technical hurdle for most laboratories in resource-limited MERS-CoV endemic countries. Here, we developed, compared and evaluated three different indirect ELISAs based on MERS-CoV nucleocapsid protein (N), spike (S) ectodomain (amino acids 1–1297) and S1 subunit (amino acids 1–725) and compared them with MN assay. The developed ELISAs were evaluated using large number of confirmed seropositive (79 samples) and seronegative (274 samples) MERS-CoV human serum samples. Both rS1- and rS-ELISAs maintained high sensitivity and specificity (≥90%) across a wider range of OD values compared to rN-ELISA. Moreover, rS1- and rS-based ELISAs showed better agreement and correlation with MN assay in contrast to rN-ELISA. Collectively, our data demonstrate that rS1-ELISA and rS-ELISA are more reliable than rN-ELISA and represent a suitable choice for seroepidemiological testing and surveillance in MERS-CoV endemic regions.

Antidiabetic plant proteins/peptides as complementary and alternative medicine – analytical perspectives

AbstractMany of the plant proteins are used as medicinal agents in the treatment of various diseases/disorders as they are produced by using molecular tools of biotechnology. Each protein is unique in its amino acid composition, sequence, subunit structures, size, shape, net charge,iso electricpoint, solubility, heat stability and hydrophobicity known to play a major role in the isolation and characterization procedures. The study of the protein of interest out of a large number is not possible unless it is obtained in its highly purified and intact form. Extraction, purification and characterization of proteins for different sample types are useful in determining structural, functional and other biological information in the field of pharmacy. Hence, the present review focuses on the sources, isolation, purification and characterization of natural proteins which are proven to be antidiabetic so as to commercialize these drugs (neutraceuticals) to compete with insulin, an ultimate in the treatment of diabetes mellitus.

Enhancement of Heat Stability and Kinetic Parameters of the Maize Endosperm ADP-Glucose Pyrophosphorylase by Mutagenesis of Amino Acids in the Small Subunit With High B Factors.

ADP-glucose pyrophosphorylase (AGPase) is an important enzyme in starch synthesis and previous studies showed that the heat lability of this enzyme is a determinant to starch synthesis in the maize endosperm and, in turn, seed yield. Here, amino acids in the AGPase endosperm small subunit with high B-factors were mutagenized and individual changes enhancing heat stability and/or kinetic parameters in an Escherichia coli expression system were chosen. Individual mutations were combined and analyzed. One triple mutant, here termed Bt2-BF, was chosen for further study. Combinations of this heat stable, 3-PGA-independent small subunit variant with large subunits also heat stable yielded complex patterns of heat stability and kinetic and allosteric properties. Interestingly, two of the three changes reside in a protein motif found only in AGPases that exhibit high sensitivity to 3-PGA. While not the 3-PGA binding site, amino acid substitutions in this region significantly alter 3-PGA activation kinetics.

Distribution and effects of natural selenium in soybean proteins and its protective role in soybean β-conglycinin (7S globulins) under AAPH-induced oxidative stress

The effects of selenium (Se) on the protein content, amino acid profile, secondary structure and subunit composition of soy proteins and its distribution were evaluated, as was the effect of peroxyl radicals produced by thermal decomposition of AAPH on the conformational changes of Se-enriched β-conglycinin (S-7S). The Se biofortification ability of soy was very strong, 7S had strongest ability to incorporate Se, and lower amounts of inorganic Se existed in Se-enriched beans. Se could promote protein synthesis and thus improve the protein content, increase the total amino acid content with a decrease in cysteine, combine into low-molecular-weight proteins, and influence the secondary structure of soybean proteins. Se was involved in the relevant protein changes in surface hydrophobicity, intrinsic fluorescence, infrared absorption and solubility and played an antioxidant role as an effectual “protector” to reduce the influence of peroxyl radical oxidation on S-7S, thereby maintaining the structural rearrangement between aggregation and protein unfolding.

The S2 glycoprotein subunit of porcine epidemic diarrhea virus contains immunodominant neutralizing epitopes

The porcine epidemic diarrhea virus (PEDV) spike (S) protein is the major target of neutralizing antibodies against PEDV. Here immunodominant neutralizing epitopes of PEDV were identified using a panel of S-specific monoclonal antibodies (mAbs). Ten of eleven S-specific mAbs successfully neutralized PEDV infectivity in vitro. Notably, epitope mapping by peptide ELISAs revealed that nine of these mAbs recognized linear neutralizing epitopes located in the N-terminus of the S2 glycoprotein subunit (amino acids [aa] 744–759, 747–774 and/or 756–771). Additionally, one mAb recognized a neutralizing epitope located in the C-terminus of S2 (aa 1371–1377), while only one neutralizing mAb reacted against a region of the S1 glycoprotein subunit (aa 499–600). Notably, mAbs that recognized epitopes within the S2 subunit presented the highest neutralizing activity against PEDV. Together these results indicate that the S2 glycoprotein subunit contains major antigenic determinants and, perhaps, the immunodominant neutralizing epitopes of PEDV.