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Sample records for lectin-like antifreeze protein

  1. Plant lectin-like antibacterial proteins from phytopathogens Pseudomonas syringae and Xanthomonas citri.

    PubMed

    Ghequire, Maarten G K; Li, Wen; Proost, Paul; Loris, Remy; De Mot, René

    2012-08-01

    The genomes of Pseudomonas syringae pv. syringae 642 and Xanthomonas citri pv. malvacearum LMG 761 each carry a putative homologue of the plant lectin-like bacteriocin (llpA) genes previously identified in the rhizosphere isolate Pseudomonas putida BW11M1 and the biocontrol strain Pseudomonas fluorescens Pf-5. The respective purified recombinant proteins, LlpAPss642 and LlpAXcm761 , display genus-specific antibacterial activity across species boundaries. The inhibitory spectrum of the P. syringae bacteriocin overlaps partially with those of the P. putida and P. fluorescens LlpAs. Notably, Xanthomonas axonopodis pv. citri str. 306 secretes a protein identical to LlpAXcm761 . The functional characterization of LlpA proteins from two different phytopathogenic γ-proteobacterial species expands the lectin-like bacteriocin family beyond the Pseudomonas genus and suggests its involvement in competition among closely related plant-associated bacteria with different lifestyles.

  2. Natural and Artificial Antifreeze Proteins

    NASA Astrophysics Data System (ADS)

    Arai, Soichi; Hirao, Noriko

    In the blood of winter polar fish an antifreeze glycoprotein (AFGP) occurs which acts to protect the fish from freezing to death. The AFGP has a unique hydrophilic hydrophobic conformation, involved in non-colligative depression of the freezing temperature of water without altering the melting point of ice. This phenomenon is reportedly a reflection of the ice crystal growth inhibition by the adsorption of the AFGP onto a-axial surfaces of the ice crystal. The authors, on the other hand, have developed an enzymatically modified protein (EMG-12) by covalent attachment of leucine dodecyl ester to the C-terminal position of gelation with the aid of a reverse reaction catalyzed by a protease. EMG-12, having a hydrophilic-hydrophobic structure, is highly surface-active and acts to stabilize a supercooling state of water by antinucleation. Discussions are made on similarities and dissimilarities of structure-function relationships of these natural and artificial antifreeze proteins. The significance of using them as antifreeze agents is also discussed.

  3. Local ice melting by an antifreeze protein.

    PubMed

    Calvaresi, Matteo; Höfinger, Siegfried; Zerbetto, Francesco

    2012-07-09

    Antifreeze proteins, AFP, impede freezing of bodily fluids and damaging of cellular tissues by low temperatures. Adsorption-inhibition mechanisms have been developed to explain their functioning. Using in silico Molecular Dynamics, we show that type I AFP can also induce melting of the local ice surface. Simulations of antifreeze-positive and antifreeze-negative mutants show a clear correlation between melting induction and antifreeze activity. The presence of local melting adds a function to type I AFPs that is unique to these proteins. It may also explain some apparently conflicting experimental results where binding to ice appears both quasipermanent and reversible.

  4. A role for a Hevea latex lectin-like protein in mediating rubber particle aggregation and latex coagulation.

    PubMed

    Wititsuwannakul, Rapepun; Pasitkul, Piyaporn; Kanokwiroon, Kamonwan; Wititsuwannakul, Dhirayos

    2008-01-01

    An in vitro aggregation of washed lutoid membrane and rubber particles, respectively, prepared from the bottom (lutoid) fraction and rubber layer of centrifuged fresh latex, leading to the formation of rubber coagulum necessary for a latex coagulation was demonstrated. A Triton X-100 extract of washed lutoid membrane proteins, isolated and prepared from the bottom fraction of centrifuged fresh latex was examined for its role in the latex coagulation process. It induced agglutination of rabbit erythrocytes, indicating the presence of a lectin-like protein. Hevea latex lectin-like protein (HLL) was purified to homogeneity by active chitin binding separation, followed by DEAE-Sepharose chromatography. Its M(r) analyzed by SDS-PAGE was 17 kDa, whereas that determined by gel filtration was 267 kDa. The HLL had a pI value of 7.2. Several glycoproteins were shown to inhibit the HLL-induced hemagglutination. The hemagglutinin activity of HLL was enhanced by Ca(2+). Of most interest was the finding that HLL strongly induced aggregation of the Hevea latex rubber particles (RP). This strong RP aggregation leads to latex coagulation, indicating the possibility that it is involved in the formation of the coagulum that plugs the latex vessel ends and stops the flow of latex upon tapping. In addition, the purified HLL also induced aggregation of RP taken from several other non-Hevea latex producing plants. This might indicate either a common or universal role of this lectin-like protein in RP aggregation and hence latex coagulation. This paper, for the first time, provides clear and unequivocal evidence for either a key biological role or physiological function of an endogenous latex lectin-like protein in the sequential process of latex coagulation.

  5. In Silico Study to Develop a Lectin-Like Protein from Mushroom Agaricus bisporus for Pharmaceutical Application

    PubMed Central

    Ismaya, Wangsa Tirta; Yunita; Damayanti, Sophi; Wijaya, Caroline; Tjandrawinata, Raymond R.; Retnoningrum, Debbie Sofie; Rachmawati, Heni

    2016-01-01

    A lectin-like protein of unknown function designated as LSMT was recently discovered in the edible mushroom Agaricus bisporus. The protein shares high structural similarity to HA-33 from Clostridium botulinum (HA33) and Ricin-B-like lectin from the mushroom Clitocybe nebularis (CNL), which have been developed as drug carrier and anti-cancer, respectively. These homologous proteins display the ability to penetrate the intestinal epithelial cell monolayer, and are beneficial for oral administration. As the characteristics of LSMT are unknown, a structural study in silico was performed to assess its potential pharmaceutical application. The study suggested potential binding to target ligands such as HA-33 and CNL although the nature, specificity, capacity, mode, and strength may differ. Further molecular docking experiments suggest that interactions between the LSMT and tested ligands may take place. This finding indicates the possible use of the LSMT protein, initiating new research on its use for pharmaceutical purposes. PMID:27110510

  6. Protein-water dynamics in antifreeze protein III activity

    NASA Astrophysics Data System (ADS)

    Xu, Yao; Bäumer, Alexander; Meister, Konrad; Bischak, Connor G.; DeVries, Arthur L.; Leitner, David M.; Havenith, Martina

    2016-03-01

    We combine Terahertz absorption spectroscopy (THz) and molecular dynamics (MD) simulations to investigate the underlying molecular mechanism for the antifreeze activity of one class of antifreeze protein, antifreeze protein type III (AFP-III) with a focus on the collective water hydrogen bond dynamics near the protein. After summarizing our previous work on AFPs, we present a new investigation of the effects of cosolutes on protein antifreeze activity by adding sodium citrate to the protein solution of AFP-III. Our results reveal that for AFP-III, unlike some other AFPs, the addition of the osmolyte sodium citrate does not affect the hydrogen bond dynamics at the protein surface significantly, as indicated by concentration dependent THz measurements. The present data, in combination with our previous THz measurements and molecular simulations, confirm that while long-range solvent perturbation is a necessary condition for the antifreeze activity of AFP-III, the local binding affinity determines the size of the hysteresis.

  7. Synthesis of Lectin-Like Protein in Developing Cotyledons of Normal and Phytohemagglutinin-Deficient Phaseolus vulgaris1

    PubMed Central

    Vitale, Alessandro; Zoppè, Monica; Fabbrini, M. Serena; Genga, Annamaria; Rivas, Liliana; Bollini, Roberto

    1989-01-01

    The genome of the common bean Phaseolus vulgaris contains a small gene family that encodes lectin and lectin-like proteins (phytohemagglutinin, arcelin, and others). One of these phytohemagglutinin-like genes was cloned by L. M. Hoffman et al. ([1982] Nucleic Acids Res 10: 7819-7828), but its product in bean cells has never been identified. We identified the product of this gene, referred to as lectin-like protein (LLP), as an abundant polypeptide synthesized on the endoplasmic reticulum (ER) of developing bean cotyledons. The gene product was first identified in extracts of Xenopus oocytes injected with either cotyledonary bean RNA or LLP-mRNA obtained by hybrid-selection with an LLP cDNA clone. A tryptic map of this protein was identical with a tryptic map of a polypeptide with the same SDS-PAGE mobility detectable in the ER of bean cotyledons pulse-labeled with either [3H]glucosamine or [3H]amino acids, both in a normal and in a phytohemagglutinin-deficient cultivar (cultivars Greensleeves and Pinto UI 111). Greensleeves LLP has Mr 40,000 and most probably has four asparagine-linked glycans. Pinto UI 111 LLP has Mr 38,500. Unlike phytohemagglutinin which is a tetramer, LLP appears to be a monomer by gel filtration analysis. Incorporation of [3H]amino acids indicates that synthesis of LLP accounts for about 3% of the proteins synthesized on the ER, a level similar to that of phytohemagglutinin. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:16666845

  8. Characterization of. alpha. -amylase-inhibitor, a lectin-like protein in the seeds of Phaseolus vulgaris

    SciTech Connect

    Moreno, J.; Altabella, T.; Chrispeels, M.J. )

    1990-03-01

    The common bean, Phaseolus vulgaris, contains a glycoprotein that inhibits the activity of mammalian and insect {alpha}-amylases but not of plant {alpha}-amylases. It is therefore classified as an antifeedant or seed defense protein. In P. vulgaris cv Greensleeves, {alpha}-amylase inhibitor ({alpha}Al) is present in embryonic axes and cotyledons, but not in other organs of the plant. The protein is synthesized during the same time period that phaseolin and phytohemagglutinin are made and also accumulates in the protein storage vacuoles (protein bodies). All the glycoforms have complex glycans that are resistant to removal by endoglycosidase H, indicating transport of the protein through the Golgi apparatus. The two different polypeptides correspond to the N-terminal and C-terminal halves of a lectin-like protein encoded by an already identified gene or a gene closely related to it. The primary translation product of {alpha}Al is a polypeptide of M{sub r} 28,000. Immunologically cross-reacting glycopolypeptides of M{sub r} 30,000 to 35,000 are present in the endoplasmic reticulum, while the smaller polypeptides (M{sub r} 15,000-19,000) accumulate in protein storage vacuoles (protein bodies). Together these data indicate that {alpha}Al is a typical bean lectin-type protein that is synthesized on the rough endoplasmic reticulum, modified in the Golgi, and transported to the protein storage vacuoles.

  9. The Structure of the Poxvirus A33 Protein Reveals a Dimer of Unique C-Type Lectin-Like Domains

    SciTech Connect

    Su, Hua-Poo; Singh, Kavita; Gittis, Apostolos G.; Garboczi, David N.

    2010-11-03

    The current vaccine against smallpox is an infectious form of vaccinia virus that has significant side effects. Alternative vaccine approaches using recombinant viral proteins are being developed. A target of subunit vaccine strategies is the poxvirus protein A33, a conserved protein in the Chordopoxvirinae subfamily of Poxviridae that is expressed on the outer viral envelope. Here we have determined the structure of the A33 ectodomain of vaccinia virus. The structure revealed C-type lectin-like domains (CTLDs) that occur as dimers in A33 crystals with five different crystal lattices. Comparison of the A33 dimer models shows that the A33 monomers have a degree of flexibility in position within the dimer. Structural comparisons show that the A33 monomer is a close match to the Link module class of CTLDs but that the A33 dimer is most similar to the natural killer (NK)-cell receptor class of CTLDs. Structural data on Link modules and NK-cell receptor-ligand complexes suggest a surface of A33 that could interact with viral or host ligands. The dimer interface is well conserved in all known A33 sequences, indicating an important role for the A33 dimer. The structure indicates how previously described A33 mutations disrupt protein folding and locates the positions of N-linked glycosylations and the epitope of a protective antibody.

  10. Characterization of α-Amylase-Inhibitor, a Lectin-Like Protein in the Seeds of Phaseolus vulgaris1

    PubMed Central

    Moreno, Joaquin; Altabella, Teresa; Chrispeels, Maarten J.

    1990-01-01

    The common bean, Phaseolus vulgaris, contains a glycoprotein that inhibits the activity of mammalian and insect α-amylases, but not of plant α-amylases. It is therefore classified as an antifeedant or seed defense protein. In P. vulgaris cv Greensleeves, α-amylase inhibitor (αAl) is present in embryonic axes and cotyledons, but not in other organs of the plant. The protein is synthesized during the same time period that phaseolin and phytohemagglutinin are made and also accumulates in the protein storage vacuoles (protein bodies). Purified αAl can be resolved by SDS-PAGE into five bands (Mr 15,000-19,000), four of which have covalently attached glycans. These bands represent glycoforms of two different polypeptides. All the glycoforms have complex glycans that are resistant to removal by endoglycosidase H, indicating transport of the protein through the Golgi apparatus. The two different polypeptides correspond to the N-terminal and C-terminal halves of a lectin-like protein encoded by an already identified gene or a gene closely related to it (LM Hoffman [1984] J Mol Appl Genet 2: 447-453; J Moreno, MJ Chrispeels [1989] Proc Natl Acad Sci USA 86:7885-7889). The primary translation product of αAl is a polypeptide of Mr 28,000. Immunologically cross-reacting glycopolypeptides of Mr 30,000 to 35,000 are present in the endoplasmic reticulum, while the smaller polypeptides (Mr 15,000-19,000) accumulate in protein storage vacuoles (protein bodies). Together these data indicate that αAl is a typical bean lectin-type protein that is synthesized on the rough endoplasmlc reticulum, modified in the Golgi, and transported to the protein storage vacuoles. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 PMID:16667338

  11. Proteins with an Euonymus lectin-like domain are ubiquitous in Embryophyta

    PubMed Central

    2009-01-01

    Background Cloning of the Euonymus lectin led to the discovery of a novel domain that also occurs in some stress-induced plant proteins. The distribution and the diversity of proteins with an Euonymus lectin (EUL) domain were investigated using detailed analysis of sequences in publicly accessible genome and transcriptome databases. Results Comprehensive in silico analyses indicate that the recently identified Euonymus europaeus lectin domain represents a conserved structural unit of a novel family of putative carbohydrate-binding proteins, which will further be referred to as the Euonymus lectin (EUL) family. The EUL domain is widespread among plants. Analysis of retrieved sequences revealed that some sequences consist of a single EUL domain linked to an unrelated N-terminal domain whereas others comprise two in tandem arrayed EUL domains. A new classification system for these lectins is proposed based on the overall domain architecture. Evolutionary relationships among the sequences with EUL domains are discussed. Conclusion The identification of the EUL family provides the first evidence for the occurrence in terrestrial plants of a highly conserved plant specific domain. The widespread distribution of the EUL domain strikingly contrasts the more limited or even narrow distribution of most other lectin domains found in plants. The apparent omnipresence of the EUL domain is indicative for a universal role of this lectin domain in plants. Although there is unambiguous evidence that several EUL domains possess carbohydrate-binding activity further research is required to corroborate the carbohydrate-binding properties of different members of the EUL family. PMID:19930663

  12. Structure and function of antifreeze proteins.

    PubMed Central

    Davies, Peter L; Baardsnes, Jason; Kuiper, Michael J; Walker, Virginia K

    2002-01-01

    High-resolution three-dimensional structures are now available for four of seven non-homologous fish and insect antifreeze proteins (AFPs). For each of these structures, the ice-binding site of the AFP has been defined by site-directed mutagenesis, and ice etching has indicated that the ice surface is bound by the AFP. A comparison of these extremely diverse ice-binding proteins shows that they have the following attributes in common. The binding sites are relatively flat and engage a substantial proportion of the protein's surface area in ice binding. They are also somewhat hydrophobic -- more so than that portion of the protein exposed to the solvent. Surface-surface complementarity appears to be the key to tight binding in which the contribution of hydrogen bonding seems to be secondary to van der Waals contacts. PMID:12171656

  13. The lectin-like protein 1 in Lactobacillus rhamnosus GR-1 mediates tissue-specific adherence to vaginal epithelium and inhibits urogenital pathogens.

    PubMed

    Petrova, Mariya I; Lievens, Elke; Verhoeven, Tine L A; Macklaim, Jean M; Gloor, Gregory; Schols, Dominique; Vanderleyden, Jos; Reid, Gregor; Lebeer, Sarah

    2016-11-21

    The probiotic Lactobacillus rhamnosus GR-1 has been documented to survive implantation onto the vaginal epithelium and interfere with urogenital pathogens. However, the molecular mechanisms involved are largely unknown. Here, we report for the first time the construction of dedicated knock-out mutants in L. rhamnosus GR-1 to enable the study of gene functions. In a search for genes responsible for the adherence capacity of L. rhamnosus GR-1, a genomic region encoding a protein with homology to lectin-like proteins was identified. Phenotypic analyses of the knock-out mutant of L. rhamnosus GR-1 revealed a two-fold decreased adhesion to the vaginal and ectocervical epithelial cell lines compared to wild-type. In contrast, the adhesion to gastro-intestinal epithelial (Caco2) and endocervical cell lines (Hela and End1/E6E7) was not drastically affected by the mutation, suggesting that the LGR-1_Llp1 lectins mediates tissue tropism. The purified LGR-1_Llp1 protein also inhibited biofilm formation and adhesion of uropathogenic Escherichia coli. For the first time, an important role for a novel lectin-like protein in the adhesion capacity and host cell-specific interaction of a vaginal probiotic Lactobacillus strain has been discovered, with an additional role in pathogen inhibition.

  14. The lectin-like protein 1 in Lactobacillus rhamnosus GR-1 mediates tissue-specific adherence to vaginal epithelium and inhibits urogenital pathogens

    PubMed Central

    Petrova, Mariya I.; Lievens, Elke; Verhoeven, Tine L. A.; Macklaim, Jean M.; Gloor, Gregory; Schols, Dominique; Vanderleyden, Jos; Reid, Gregor; Lebeer, Sarah

    2016-01-01

    The probiotic Lactobacillus rhamnosus GR-1 has been documented to survive implantation onto the vaginal epithelium and interfere with urogenital pathogens. However, the molecular mechanisms involved are largely unknown. Here, we report for the first time the construction of dedicated knock-out mutants in L. rhamnosus GR-1 to enable the study of gene functions. In a search for genes responsible for the adherence capacity of L. rhamnosus GR-1, a genomic region encoding a protein with homology to lectin-like proteins was identified. Phenotypic analyses of the knock-out mutant of L. rhamnosus GR-1 revealed a two-fold decreased adhesion to the vaginal and ectocervical epithelial cell lines compared to wild-type. In contrast, the adhesion to gastro-intestinal epithelial (Caco2) and endocervical cell lines (Hela and End1/E6E7) was not drastically affected by the mutation, suggesting that the LGR-1_Llp1 lectins mediates tissue tropism. The purified LGR-1_Llp1 protein also inhibited biofilm formation and adhesion of uropathogenic Escherichia coli. For the first time, an important role for a novel lectin-like protein in the adhesion capacity and host cell-specific interaction of a vaginal probiotic Lactobacillus strain has been discovered, with an additional role in pathogen inhibition. PMID:27869151

  15. Fish antifreeze protein and the freezing and recrystallization of ice.

    PubMed

    Knight, C A; DeVries, A L; Oolman, L D

    Antifreeze glycopeptide and peptides from the blood of polar fishes prevent the growth of ice crystals in water at temperatures down to approximately 1 degree C below freezing point, but do not appreciably influence the equilibrium freezing point. This freezing point hysteresis must be a disequilibrium effect, or it would violate Gibbs' phase rule, but the separate freezing and melting points are experimentally very definite: ice neither melts nor freezes perceptibly within the 'hysteresis gap', for periods of hours or days. We report here unusual crystal faces on ice crystals grown from solutions of very low concentrations of the anti-freeze glycopeptides and peptides. This is a clue to the mechanism of freezing inhibition, and it may be the basis of a simple, very sensitive test for antifreeze material. Very low concentrations of the antifreeze protein are also remarkably effective in preventing the recrystallization of ice.

  16. Lebecin, a new C-type lectin like protein from Macrovipera lebetina venom with anti-tumor activity against the breast cancer cell line MDA-MB231.

    PubMed

    Jebali, Jed; Fakhfekh, Emna; Morgen, Maram; Srairi-Abid, Najet; Majdoub, Hafedh; Gargouri, Ali; El Ayeb, Mohamed; Luis, José; Marrakchi, Naziha; Sarray, Sameh

    2014-08-01

    C-type lectins like proteins display various biological activities and are known to affect especially platelet aggregation. Few of them have been reported to have anti-tumor effects. In this study, we have identified and characterized a new C-type lectin like protein, named lebecin. Lebecin is a heterodimeric protein of 30 kDa. The N-terminal amino acid sequences of both subunits were determined by Edman degradation and the entire amino acid sequences were deduced from cDNAs. The precursors of both lebecin subunits contain a 23-amino acid residue signal peptide and the mature α and β subunits are composed of 129 and 131 amino acids, respectively. Lebecin is shown to be a potent inhibitor of MDA-MB231 human breast cancer cells proliferation. Furthermore, lebecin dose-dependently inhibited the integrin-mediated attachment of these cells to different adhesion substrata. This novel C-type lectin also completely blocked MDA-MB231 cells migration towards fibronectin and fibrinogen in haptotaxis assays.

  17. Properties, potentials, and prospects of antifreeze proteins.

    PubMed

    Venketesh, S; Dayananda, C

    2008-01-01

    Antifreeze proteins (AFPs) are a group of proteins that protect organisms from deep freezing temperatures and are expressed in vertebrates, invertebrates, plants, bacteria, and fungi. The nuclear magnetic resonance, x-ray structure, and many spectroscopic studies with AFPs have been instrumental in determining the structure-function relationship. Mutational studies have indicated the importance of hydrophobic residues in ice binding. Various studies have pointed out that the mechanism of AFP action is through its adsorption on the ice surface, which leads to a curved surface, preventing further growth of ice by the "Kelvin effect." The AFPs have potential industrial, medical, and agricultural application in different fields, such as food technology, preservation of cell lines, organs, cryosurgery, and cold hardy transgenic plants and animals. However, the applications of AFPs are marred by high cost due to low yield. This review deals with the source and properties of AFPs from an angle of their application and their potential. The possibility of production using different molecular biological techniques, which will help increase the yield, is also dealt with.

  18. Type I Antifreeze Proteins Enhance Ice Nucleation above Certain Concentrations

    PubMed Central

    Wilson, Peter W.; Osterday, Katie E.; Heneghan, Aaron F.; Haymet, Anthony D. J.

    2010-01-01

    In this study, we examined the effects that antifreeze proteins have on the supercooling and ice-nucleating abilities of aqueous solutions. Very little information on such nucleation currently exists. Using an automated lag time apparatus and a new analysis, we show several dilution series of Type I antifreeze proteins. Our results indicate that, above a concentration of ∼8 mg/ml, ice nucleation is enhanced rather than hindered. We discuss this unexpected result and present a new hypothesis outlining three components of polar fish blood that we believe affect its solution properties in certain situations. PMID:20837472

  19. Molecular modeling of lectin-like protein from Acacia farnesiana reveals a possible anti-inflammatory mechanism in Carrageenan-induced inflammation.

    PubMed

    Abrantes, Vanessa Erika Ferreira; Matias da Rocha, Bruno Anderson; Batista da Nóbrega, Raphael; Silva-Filho, José Caetano; Teixeira, Claudener Souza; Cavada, Benildo Sousa; Gadelha, Carlos Alberto de Almeida; Ferreira, Sergio Henrique; Figueiredo, Jozi Godoy; Santi-Gadelha, Tatiane; Delatorre, Plinio

    2013-01-01

    Acacia farnesiana lectin-like protein (AFAL) is a chitin-binding protein and has been classified as phytohaemagglutinin from Phaseolus vulgaris (PHA). Legume lectins are examples for structural studies, and this family of proteins shows a remarkable conservation in primary, secondary, and tertiary structures. Lectins have ability to reduce the effects of inflammation caused by phlogistic agents, such as carrageenan (CGN). This paper explains the anti-inflammatory activity of AFAL through structural comparison with anti-inflammatory legume lectins. The AFAL model was obtained by molecular modeling and molecular docking with glycan and carrageenan were performed to explain the AFAL structural behavior and biological activity. Pisum sativum lectin was the best template for molecular modeling. The AFAL structure model is folded as a β sandwich. The model differs from template in loop regions, number of β strands and carbohydrate-binding site. Carrageenan and glycan bind to different sites on AFAL. The ability of AFAL binding to carrageenan can be explained by absence of the sixth β -strand (posterior β sheets) and two β strands in frontal region. AFAL can inhibit pathway inflammatory process by carrageenan injection by connecting to it and preventing its entry into the cell and triggers the reaction.

  20. Characterization and sugar-binding properties of arcelin-1, an insecticidal lectin-like protein isolated from kidney bean (Phaseolus vulgaris L. cv. RAZ-2) seeds.

    PubMed Central

    Fabre, C; Causse, H; Mourey, L; Koninkx, J; Rivière, M; Hendriks, H; Puzo, G; Samama, J P; Rougé, P

    1998-01-01

    Arcelin-1 is a lectin-like protein found in the seeds of wild varieties of the kidney bean (Phaseolus vulgaris). This protein displays insecticidal properties, but the mechanism of action is as yet unknown. In the present study we investigated the biochemical and biophysical properties of arcelin-1 from Phaseolus vulgaris cv. RAZ-2. Native arcelin-1 is a dimeric glycoprotein of 60 kDa, built from the non-covalent association of two identical monomers. This dimer resists dissociation by chaotropic agents and is highly resistant to proteolytic enzymes. Each subunit contains 10% (w/w) neutral sugars which belong to the high-mannose and complex-type glycans attached to three glycosylation sites. No interaction of the protein with simple sugars could be detected, but arcelin-1 displays an intrinsic specificity in binding complex glycans. Arcelin-1 therefore differs from the closely related phytohaemagglutinin lectins and alpha-amylase inhibitor in several respects: oligomerization states, sugar-binding affinities and the type and number of glycan chains. These features may be related to the toxicity of arcelin-1. PMID:9445382

  1. The structure of the cysteine protease and lectin-like domains of Cwp84, a surface layer-associated protein from Clostridium difficile

    SciTech Connect

    Bradshaw, William J.; Kirby, Jonathan M.; Thiyagarajan, Nethaji; Chambers, Christopher J.; Davies, Abigail H.; Roberts, April K.; Shone, Clifford C.; Acharya, K. Ravi

    2014-07-01

    The crystal structure of Cwp84, an S-layer protein from Clostridium difficile is presented for the first time. The cathepsin L-like fold of cysteine protease domain, a newly observed ‘lectin-like’ domain and several other features are described. Clostridium difficile is a major problem as an aetiological agent for antibiotic-associated diarrhoea. The mechanism by which the bacterium colonizes the gut during infection is poorly understood, but undoubtedly involves a myriad of components present on the bacterial surface. The mechanism of C. difficile surface-layer (S-layer) biogenesis is also largely unknown but involves the post-translational cleavage of a single polypeptide (surface-layer protein A; SlpA) into low- and high-molecular-weight subunits by Cwp84, a surface-located cysteine protease. Here, the first crystal structure of the surface protein Cwp84 is described at 1.4 Å resolution and the key structural components are identified. The truncated Cwp84 active-site mutant (amino-acid residues 33–497; C116A) exhibits three regions: a cleavable propeptide and a cysteine protease domain which exhibits a cathepsin L-like fold followed by a newly identified putative carbohydrate-binding domain with a bound calcium ion, which is referred to here as a lectin-like domain. This study thus provides the first structural insights into Cwp84 and a strong base to elucidate its role in the C. difficile S-layer maturation mechanism.

  2. Molecular Modeling of Lectin-Like Protein from Acacia farnesiana Reveals a Possible Anti-Inflammatory Mechanism in Carrageenan-Induced Inflammation

    PubMed Central

    Abrantes, Vanessa Erika Ferreira; Matias da Rocha, Bruno Anderson; Batista da Nóbrega, Raphael; Silva-Filho, José Caetano; Teixeira, Claudener Souza; Cavada, Benildo Sousa; Gadelha, Carlos Alberto de Almeida; Ferreira, Sergio Henrique; Figueiredo, Jozi Godoy; Santi-Gadelha, Tatiane; Delatorre, Plinio

    2013-01-01

    Acacia farnesiana lectin-like protein (AFAL) is a chitin-binding protein and has been classified as phytohaemagglutinin from Phaseolus vulgaris (PHA). Legume lectins are examples for structural studies, and this family of proteins shows a remarkable conservation in primary, secondary, and tertiary structures. Lectins have ability to reduce the effects of inflammation caused by phlogistic agents, such as carrageenan (CGN). This paper explains the anti-inflammatory activity of AFAL through structural comparison with anti-inflammatory legume lectins. The AFAL model was obtained by molecular modeling and molecular docking with glycan and carrageenan were performed to explain the AFAL structural behavior and biological activity. Pisum sativum lectin was the best template for molecular modeling. The AFAL structure model is folded as a β sandwich. The model differs from template in loop regions, number of β strands and carbohydrate-binding site. Carrageenan and glycan bind to different sites on AFAL. The ability of AFAL binding to carrageenan can be explained by absence of the sixth β-strand (posterior β sheets) and two β strands in frontal region. AFAL can inhibit pathway inflammatory process by carrageenan injection by connecting to it and preventing its entry into the cell and triggers the reaction. PMID:24490151

  3. The antifreeze potential of the spruce budworm thermal hysteresis protein.

    PubMed

    Tyshenko, M G; Doucet, D; Davies, P L; Walker, V K

    1997-09-01

    Antifreeze proteins (AFP) inhibit ice growth by surface adsorption that results in a depression of the freezing point below the melting point. The maximum level of this thermal hysteresis shown by the four structurally unrelated fish AFP is approximately 1.5 degrees C. In contrast, hemolymph and crude extracts from insects can have 5 degrees to 10 degrees C of thermal hysteresis. Based on the isolation, cloning, and expression of a thermal hysteresis protein (THP) from spruce budworm (Choristoneura fumiferana), the vastly greater activity is attributable to a 9 kDa protein. This novel, threonine- and cysteine-rich THP has striking effects on ice crystal morphology, both before and during freezing. It is also 10 to 30 times more active than any known fish AFP, offering the prospect of superior antifreeze properties in cryoprotective applications.

  4. Overexpression of L-type lectin-like protein kinase 1 confers pathogen resistance and regulates salinity response in Arabidopsis thaliana.

    PubMed

    Huang, Ping; Ju, Hyun-Woo; Min, Ji-Hee; Zhang, Xia; Kim, Su-Hyun; Yang, Kwang-Yeol; Kim, Cheol Soo

    2013-04-01

    Plant receptor-like protein kinases are thought to be involved in various cellular processes mediated by signal transduction pathways. There are about 45 lectin receptor kinases in Arabidopsis, but only a few have been studied. Here, we investigated the effect of the disruption and overexpression of a plasma membrane-localized L-type lectin-like protein kinase 1, AtLPK1 (At4g02410), on plant responses to abiotic and biotic stress. Expression of AtLPK1 was strongly induced by abscisic acid, methyl jasmonate, salicylic acid and stress treatments. Overexpression of AtLPK1 in Arabidopsis resulted in enhanced seed germination and cotyledon greening under high salinity condition, while antisense transgenic lines were more sensitive to salt stress. Activity of three abiotic stress responsive genes, RD29A, RD29B and COR15A, was elevated in AtLPK1-overexpressing plants than that in wild type (WT) plants with salt treatment, whereas the transcript level of these genes in antisense plants decreased compared with WT. Furthermore, AtLPK1-overexpressing plants displayed increased resistance to infection by Botrytis cinerea and exhibited stronger expression of a group of defense-related genes than did WT. The data implicates AtLPK1 plays essential roles at both abiotic and biotic stress response in Arabidopsis thaliana.

  5. A serendipitous discovery of antifreeze protein-specific activity in C-linked antifreeze glycoprotein analogs.

    PubMed

    Eniade, Adewale; Purushotham, Madhusudhan; Ben, Robert N; Wang, J B; Horwath, Kathleen

    2003-01-01

    Structurally diverse carbon-linked (C-linked) analogs of antifreeze glycoprotein (AFGP) have been prepared via linear or convergent solid phase synthesis. These analogs range in molecular weight from approx 1.5-4.1 KDa and do not possess the beta-D-galactose-1,3-alpha-D-N-acetylgalactosamine carbohydrate moiety or the L-threonine-L-alanine-L-alanine polypeptide backbone native to the AFGP wild-type. Despite these dramatic structural modifications, the 2.7-KDa and 4.1-KDa analogs possess antifreeze protein-specific activity as determined by recrystallization-inhibition (RI) and thermal hysteresis (TH) assays. These analogs are weaker than the wild-type in their activity, but nanoliter osmometry indicates that these compounds are binding to ice and affecting a localized freezing point depression. This is the first example of a C-linked AFGP analog that possesses TH and RI activity and suggests that the rational design and synthesis of chemically and biologically stable AFGP analogs is a feasible and worthwhile endeavor. Given the low degree of TH activity, these compounds may prove useful for the protection of cells during freezing and thawing cycles.

  6. Anti-virulence properties of an antifreeze protein

    PubMed Central

    Heisig, Martin; Abraham, Nabil M.; Liu, Lei; Neelakanta, Girish; Mattessich, Sarah; Sultana, Hameeda; Shang, Zhengling; Ansari, Juliana M.; Killiam, Charlotte; Walker, Wendy; Cooley, Lynn; Flavell, Richard A.; Agaisse, Herve; Fikrig, Erol

    2014-01-01

    Summary As microbial drug-resistance increases, there is a critical need for new classes of compounds to combat infectious diseases. The Ixodes scapularis tick antifreeze glycoprotein, IAFGP, functions as an anti-virulence agent against diverse bacteria including methicillin-resistant Staphylococcus aureus. Recombinant IAFGP and a peptide, P1, derived from this protein bind to microbes and alter biofilm formation. Transgenic iafgp-expressing flies and mice challenged with bacteria, as well as wild-type animals administered P1, were resistant to infection, septic shock, or biofilm development on implanted catheter tubing. These data show that an antifreeze protein facilitates host control of bacterial infections and suggest new therapeutic strategies to counter pathogens. PMID:25373896

  7. Hexagonal shaped ice spicules in frozen antifreeze protein solutions.

    PubMed

    Wilson, P W; Gould, M; DeVries, A L

    2002-06-01

    In the presence of antifreeze proteins from both Antarctic and Arctic fishes, water freezes in the form of long c-axis spikes or spicular-like crystals. Transmission electron microscopy of the Pt/C replicas of the freeze fractured spicular ice in a small capillary revealed the presence of many hexagonally shaped structures whose cross-sectional dimensions were between 0.5 and 10 microm. Well-defined parallel faces were associated with most fractured and etched spicules. When fracture planes occurred near the tip of a spicule, well-defined pyramidal faces were apparent. Steps were sometimes associated with these pyramidal spicular crystal faces. On some of the replicas obvious roughening of certain crystal faces of the spicule was observed, suggesting that the antifreeze proteins may have adsorbed to those faces.

  8. Analysis of antifreeze protein activity using colorimetric gold nanosensors

    NASA Astrophysics Data System (ADS)

    Jing, Xu; Choi, Ho-seok; Park, Ji-In; Kim, Young-Pil

    2015-07-01

    High activity and long stability of antifreeze proteins (AFPs), also known as ice-binding proteins (IBPs), are necessary for exerting their physiological functions in biotechnology and cryomedicine. Here we report a simple analysis of antifreeze protein activity and stability based on self-assembly of gold nanoparticles (AuNPs) via freezing and thawing cycles. While the mercaptosuccinic acid-capped AuNP (MSA-AuNP) was easily self-assembled after a freezing/thawing cycle, due to the mechanical attack of ice crystal on the MSA-AuNP surface, the presence of AFP impeded the self-assembly of MSA-AuNP via the interaction of AFP with ice crystals via freezing and thawing cycles, which led to a strong color in the MSA-AuNP solution. As a result, the aggregation parameter (E520/E650) of MSA-AuNP showed the rapid detection of both activity and stability of AFPs. We suggest that our newly developed method is very suitable for measuring antifreeze activity and stability in a simple and rapid manner with reliable quantification.

  9. Contrasting Roles of the Apoplastic Aspartyl Protease APOPLASTIC, ENHANCED DISEASE SUSCEPTIBILITY1-DEPENDENT1 and LEGUME LECTIN-LIKE PROTEIN1 in Arabidopsis Systemic Acquired Resistance.

    PubMed

    Breitenbach, Heiko H; Wenig, Marion; Wittek, Finni; Jordá, Lucia; Maldonado-Alconada, Ana M; Sarioglu, Hakan; Colby, Thomas; Knappe, Claudia; Bichlmeier, Marlies; Pabst, Elisabeth; Mackey, David; Parker, Jane E; Vlot, A Corina

    2014-06-01

    Systemic acquired resistance (SAR) is an inducible immune response that depends on ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1). Here, we show that Arabidopsis (Arabidopsis thaliana) EDS1 is required for both SAR signal generation in primary infected leaves and SAR signal perception in systemic uninfected tissues. In contrast to SAR signal generation, local resistance remains intact in eds1 mutant plants in response to Pseudomonas syringae delivering the effector protein AvrRpm1. We utilized the SAR-specific phenotype of the eds1 mutant to identify new SAR regulatory proteins in plants conditionally expressing AvrRpm1. Comparative proteomic analysis of apoplast-enriched extracts from AvrRpm1-expressing wild-type and eds1 mutant plants led to the identification of 12 APOPLASTIC, EDS1-DEPENDENT (AED) proteins. The genes encoding AED1, a predicted aspartyl protease, and another AED, LEGUME LECTIN-LIKE PROTEIN1 (LLP1), were induced locally and systemically during SAR signaling and locally by salicylic acid (SA) or its functional analog, benzo 1,2,3-thiadiazole-7-carbothioic acid S-methyl ester. Because conditional overaccumulation of AED1-hemagglutinin inhibited SA-induced resistance and SAR but not local resistance, the data suggest that AED1 is part of a homeostatic feedback mechanism regulating systemic immunity. In llp1 mutant plants, SAR was compromised, whereas the local resistance that is normally associated with EDS1 and SA as well as responses to exogenous SA appeared largely unaffected. Together, these data indicate that LLP1 promotes systemic rather than local immunity, possibly in parallel with SA. Our analysis reveals new positive and negative components of SAR and reinforces the notion that SAR represents a distinct phase of plant immunity beyond local resistance.

  10. Basal Plane Affinity of an Insect Antifreeze Protein

    NASA Astrophysics Data System (ADS)

    Pertaya, N.; Gauthier, S. Y.; Davies, P. L.; Braslavsky, I.

    2007-03-01

    sbwAFP is a powerful antifreeze protein (AFP) with high thermal hysteresis activity that protects spruce budworm (sbw) from freezing during harsh winters in the spruce and fir forests of USA and Canada. Different types of antifreeze proteins have been found in many other species and have potential applications in cryomedicine and cryopreservation. When an ice crystal is cooled in the presence of AFP below the non-equilibrium freezing point the crystal will suddenly and rapidly grow in specific directions. Hyperactive antifreezes like sbwAFP expand perpendicular to the c-axis (in the plane of the a-axes), whereas moderately active AFPs, like type III from fish, grow in the direction parallel to the c-axis. It has been proposed that the basis for hyperactivity of certain AFPs is that they bind and accumulate on the basal plane to inhibit c-axial growth. By putting fluorescent tags on these two types of AFPs we have been able to directly visualize the binding of different types of AFPs to ice surfaces. We do indeed find that the insect AFP accumulates on the basal plane of an ice crystal while type III AFP does not. Supported by CIHR and BNTI.

  11. Pouterin, a novel potential cytotoxic lectin-like protein with apoptosis-inducing activity in tumorigenic mammalian cells.

    PubMed

    Boleti, Ana Paula de A; Ventura, Cláudio A; Justo, Giselle Z; Silva, Rodrigo A; de Sousa, Ana Carolina T; Ferreira, Carmen V; Yano, Tomomasa; Macedo, Maria Lígia R

    2008-06-15

    In this study, the cytotoxicity of pouterin in tumorigenic and non-tumorigenic mammalian cell lines was investigated. We found that HeLa, Hep-2 and HT-29 tumor cells were highly sensitive to pouterin cytotoxicity in a dose-dependent manner, whereas non-tumorigenic Vero cells and human lymphocytes were relatively resistant to the protein. Among the tumor cell lines, HeLa cells showed the highest susceptibility to pouterin cytotoxicity, exhibiting a time-dependent increase in LDH leakage and an IC(50) value of 5mug/mL. Morphological alterations such as rounding, cell shrinkage and chromatin condensation, consistent with apoptotic cell death were observed. Apoptosis induction was demonstrated by DNA fragmentation as detected by terminal dUTP nick-end labeling (TUNEL). Furthermore, HeLa cells incubated with pouterin showed disruption of the actin cytoskeleton. Western blot analysis revealed that pouterin caused increased expression of p21, thus indicating cell cycle arrest. Subsequent studies provided evidence that apoptosis may be partially explained in the activation of the tumor necrosis factor receptor 1 (TNFR1) signaling. Interestingly, a time-dependent decrease of the expression of p65 nuclear factor kappa B (NFkappaB) subunit, concomitant with a downregulation of the inhibitor of apoptosis protein 1 (IAP1) was observed, suggesting that TNFR-mediated apoptosis is the predominant pathway induced by pouterin in HeLa cells.

  12. Gram-positive bacteria are held at a distance in the colon mucus by the lectin-like protein ZG16

    PubMed Central

    Bergström, Joakim H.; Katona, Gergely; Schütte, André; Ermund, Anna; Hansson, Gunnar C.

    2016-01-01

    The distal colon functions as a bioreactor and harbors an enormous amount of bacteria in a mutualistic relationship with the host. The microbiota have to be kept at a safe distance to prevent inflammation, something that is achieved by a dense inner mucus layer that lines the epithelial cells. The large polymeric nets made up by the heavily O-glycosylated MUC2 mucin forms this physical barrier. Proteomic analyses of mucus have identified the lectin-like protein ZG16 (zymogen granulae protein 16) as an abundant mucus component. To elucidate the function of ZG16, we generated recombinant ZG16 and studied Zg16−/− mice. ZG16 bound to and aggregated Gram-positive bacteria via binding to the bacterial cell wall peptidoglycan. Zg16−/− mice have a distal colon mucus layer with normal thickness, but with bacteria closer to the epithelium. Using distal colon explants mounted in a horizontal perfusion chamber we demonstrated that treatment of bacteria with recombinant ZG16 hindered bacterial penetration into the mucus. The inner colon mucus of Zg16−/− animals had a higher load of Gram-positive bacteria and showed bacteria with higher motility in the mucus close to the host epithelium compared with cohoused littermate Zg16+/+. The more penetrable Zg16−/− mucus allowed Gram-positive bacteria to translocate to systemic tissues. Viable bacteria were found in spleen and were associated with increased abdominal fat pad mass in Zg16−/− animals. The function of ZG16 reveals a mechanism for keeping bacteria further away from the host colon epithelium. PMID:27849619

  13. Thermolabile antifreeze protein produced in Escherichia coli for structural analysis.

    PubMed

    Lin, Feng-Hsu; Sun, Tianjun; Fletcher, Garth L; Davies, Peter L

    2012-03-01

    The only hyperactive antifreeze protein (AFP) found to date in fishes is an extreme variant of the 3-kDa, alpha-helical, alanine-rich type I AFP, which is referred to here as type Ih. Purification of the 33-kDa homodimeric AFP Ih from a natural source was hampered by its low levels in fish plasma; by the need to remove the more abundant smaller isoforms; and by its extreme thermolability. Moreover, ice affinity as a purification tool was spoiled by the tendency of fish IgM antibodies to bind to ice in the presence of AFPs. In order to produce enough protein for crystallography we expressed AFP Ih as a recombinant protein in the Arctic Express® strain of Escherichia coli at 12 °C, just below the thermal denaturation temperature of 16-18 °C. His-tags were not useful because they compromised the activity and yield of AFP Ih. But in the absence of fish antibodies we were able to recover 10-mg quantities of the antifreeze protein using two cycles of ice affinity purification followed by anion-exchange chromatography to remove contaminating chaperones. The purified recombinant AFP Ih yielded diffraction-quality crystals with an extremely asymmetrical unit cell. By transferring the genes of the chaperones into a methionine auxotroph we were able to grow this host at low temperatures and produce sufficient selenomethionine-labeled AFP Ih for crystallography.

  14. Utilizing avidity to improve antifreeze protein activity: a type III antifreeze protein trimer exhibits increased thermal hysteresis activity.

    PubMed

    Can, Özge; Holland, Nolan B

    2013-12-03

    Antifreeze proteins (AFPs) are ice growth inhibitors that allow the survival of several species living at temperatures colder than the freezing point of their bodily fluids. AFP activity is commonly defined in terms of thermal hysteresis, which is the difference observed for the solution freezing and melting temperatures. Increasing the thermal hysteresis activity of these proteins, particularly at low concentrations, is of great interest because of their wide range of potential applications. In this study, we have designed and expressed one-, two-, and three-domain antifreeze proteins to improve thermal hysteresis activity through increased binding avidity. The three-domain type III AFP yielded significantly greater activity than the one- and two-domain proteins, reaching a thermal hysteresis of >1.6 °C at a concentration of <1 mM. To elucidate the basis of this increase, the data were fit to a multidomain protein adsorption model based on the classical Langmuir isotherm. Fits of the data to the modified isotherms yield values for the equilibrium binding constants for the adsorption of AFP to ice and indicate that protein surface coverage is proportional to thermal hysteresis activity.

  15. Structure and dynamics of a beta-helical antifreeze protein.

    PubMed

    Daley, Margaret E; Spyracopoulos, Leo; Jia, Zongchao; Davies, Peter L; Sykes, Brian D

    2002-04-30

    Antifreeze proteins (AFPs) protect many types of organisms from damage caused by freezing. They do this by binding to the ice surface, which causes inhibition of ice crystal growth. However, the molecular mechanism of ice binding leading to growth inhibition is not well understood. In this paper, we present the solution structure and backbone NMR relaxation data of the antifreeze protein from the yellow mealworm beetle Tenebrio molitor (TmAFP) to study the dynamics in the context of structure. The full (15)N relaxation analysis was completed at two magnetic field strengths, 500 and 600 MHz, as well as at two temperatures, 30 and 5 degrees C, to measure the dynamic changes that occur in the protein backbone at different temperatures. TmAFP is a small, highly disulfide-bonded, right-handed parallel beta-helix consisting of seven tandemly repeated 12-amino acid loops. The backbone relaxation data displays a periodic pattern, which reflects both the 12-amino acid structural repeat and the highly anisotropic nature of the protein. Analysis of the (15)N relaxation parameters shows that TmAFP is a well-defined, rigid structure, and the extracted parameters show that there is similar restricted internal mobility throughout the protein backbone at both temperatures studied. We conclude that the hydrophobic, rigid binding site may reduce the entropic penalty for the binding of the protein to ice. The beta-helical fold of the protein provides this rigidity, as it does not appear to be a consequence of cooling toward a physiologically relevant temperature.

  16. Superheating of ice crystals in antifreeze protein solutions.

    PubMed

    Celik, Yeliz; Graham, Laurie A; Mok, Yee-Foong; Bar, Maya; Davies, Peter L; Braslavsky, Ido

    2010-03-23

    It has been argued that for antifreeze proteins (AFPs) to stop ice crystal growth, they must irreversibly bind to the ice surface. Surface-adsorbed AFPs should also prevent ice from melting, but to date this has been demonstrated only in a qualitative manner. Here we present the first quantitative measurements of superheating of ice in AFP solutions. Superheated ice crystals were stable for hours above their equilibrium melting point, and the maximum superheating obtained was 0.44 degrees C. When melting commenced in this superheated regime, rapid melting of the crystals from a point on the surface was observed. This increase in melting temperature was more appreciable for hyperactive AFPs compared to the AFPs with moderate antifreeze activity. For each of the AFP solutions that exhibited superheating, the enhancement of the melting temperature was far smaller than the depression of the freezing temperature. The present findings clearly show that AFPs adsorb to ice surfaces as part of their mechanism of action, and this absorption leads to protection of ice against melting as well as freezing.

  17. Biophysical and biochemical aspects of antifreeze proteins: Using computational tools to extract atomistic information.

    PubMed

    Kar, Rajiv K; Bhunia, Anirban

    2015-11-01

    Antifreeze proteins (AFPs) are the key biomolecules that protect species from extreme climatic conditions. Studies of AFPs, which are based on recognition of ice plane and structural motifs, have provided vital information that point towards the mechanism responsible for executing antifreeze activity. Importantly, the use of experimental techniques has revealed key information for AFPs, but the exact microscopic details are still not well understood, which limits the application and design of novel antifreeze agents. The present review focuses on the importance of computational tools for investigating (i) molecular properties, (ii) structure-function relationships, and (iii) AFP-ice interactions at atomistic levels. In this context, important details pertaining to the methodological approaches used in molecular dynamics studies of AFPs are also discussed. It is hoped that the information presented herein is helpful for enriching our knowledge of antifreeze properties, which can potentially pave the way for the successful design of novel antifreeze biomolecular agents.

  18. Using support vector machine and evolutionary profiles to predict antifreeze protein sequences.

    PubMed

    Zhao, Xiaowei; Ma, Zhiqiang; Yin, Minghao

    2012-01-01

    Antifreeze proteins (AFPs) are ice-binding proteins. Accurate identification of new AFPs is important in understanding ice-protein interactions and creating novel ice-binding domains in other proteins. In this paper, an accurate method, called AFP_PSSM, has been developed for predicting antifreeze proteins using a support vector machine (SVM) and position specific scoring matrix (PSSM) profiles. This is the first study in which evolutionary information in the form of PSSM profiles has been successfully used for predicting antifreeze proteins. Tested by 10-fold cross validation and independent test, the accuracy of the proposed method reaches 82.67% for the training dataset and 93.01% for the testing dataset, respectively. These results indicate that our predictor is a useful tool for predicting antifreeze proteins. A web server (AFP_PSSM) that implements the proposed predictor is freely available.

  19. Interfacial Adsorption of Antifreeze Proteins: A Neutron Reflection Study

    PubMed Central

    Xu, Hai; Perumal, Shiamalee; Zhao, Xiubo; Du, Ning; Liu, Xiang-Yang; Jia, Zongchao; Lu, Jian R.

    2008-01-01

    Interfacial adsorption from two antifreeze proteins (AFP) from ocean pout (Macrozoarces americanus, type III AFP, AFP III, or maAFP) and spruce budworm (Choristoneura fumiferana, isoform 501, or cfAFP) were studied by neutron reflection. Hydrophilic silicon oxide was used as model substrate to facilitate the solid/liquid interfacial measurement so that the structural features from AFP adsorption can be examined. All adsorbed layers from AFP III could be modeled into uniform layer distribution assuming that the protein molecules were adsorbed with their ice-binding surface in direct contact with the SiO2 substrate. The layer thickness of 32 Å was consistent with the height of the molecule in its crystalline form. With the concentration decreasing from 2 mg/ml to 0.01 mg/ml, the volume fraction of the protein packed in the monolayer decreased steadily from 0.4 to 0.1, consistent with the concentration-dependent inhibition of ice growth observed over the range. In comparison, insect cfAFP showed stronger adsorption over the same concentration range. Below 0.1 mg/ml, uniform layers were formed. But above 1 mg/ml, the adsorbed layers were characterized by a dense middle layer and two outer diffuse layers, with a total thickness around 100 Å. The structural transition indicated the responsive changes of conformational orientation to increasing surface packing density. As the higher interfacial adsorption of cfAFP was strongly correlated with the greater thermal hysteresis of spruce budworm, our results indicated the important relation between protein adsorption and antifreeze activity. PMID:18234809

  20. Effects of polyhydroxy compounds on beetle antifreeze protein activity

    PubMed Central

    Amornwittawat, Natapol; Wang, Sen; Banatlao, Joseph; Chung, Melody; Velasco, Efrain; Duman, John G.; Wen, Xin

    2016-01-01

    Antifreeze proteins (AFPs) noncolligatively depress the nonequilibrium freezing point of a solution and produce a difference between the melting and freezing points termed thermal hysteresis (TH). Some low-molecular-mass solutes can affect the TH values. The TH enhancement effects of selected polyhydroxy compounds including polyols and carbohydrates on an AFP from the beetle Dendroides canadensis were systematically investigated using differential scanning calorimetry (DSC). The number of hydroxyl groups dominates the molar enhancement effectiveness of polyhydroxy compounds having one to five hydroxyl groups. However, the above rule does not apply for polyhydroxy compounds having more than five hydroxyl groups. The most efficient polyhydroxy enhancer identified is trehalose. In a combination of enhancers the strongest enhancer plays the major role in determining the TH enhancement. Mechanistic insights into identification of highly efficient AFP enhancers are discussed. PMID:19038370

  1. Towards a green hydrate inhibitor: imaging antifreeze proteins on clathrates.

    PubMed

    Gordienko, Raimond; Ohno, Hiroshi; Singh, Vinay K; Jia, Zongchao; Ripmeester, John A; Walker, Virginia K

    2010-02-11

    The formation of hydrate plugs in oil and gas pipelines is a serious industrial problem and recently there has been an increased interest in the use of alternative hydrate inhibitors as substitutes for thermodynamic inhibitors like methanol. We show here that antifreeze proteins (AFPs) possess the ability to modify structure II (sII) tetrahydrofuran (THF) hydrate crystal morphologies by adhering to the hydrate surface and inhibiting growth in a similar fashion to the kinetic inhibitor poly-N-vinylpyrrolidone (PVP). The effects of AFPs on the formation and growth rate of high-pressure sII gas mix hydrate demonstrated that AFPs are superior hydrate inhibitors compared to PVP. These results indicate that AFPs may be suitable for the study of new inhibitor systems and represent an important step towards the development of biologically-based hydrate inhibitors.

  2. Towards a Green Hydrate Inhibitor: Imaging Antifreeze Proteins on Clathrates

    PubMed Central

    Gordienko, Raimond; Ohno, Hiroshi; Singh, Vinay K.; Jia, Zongchao; Ripmeester, John A.; Walker, Virginia K.

    2010-01-01

    The formation of hydrate plugs in oil and gas pipelines is a serious industrial problem and recently there has been an increased interest in the use of alternative hydrate inhibitors as substitutes for thermodynamic inhibitors like methanol. We show here that antifreeze proteins (AFPs) possess the ability to modify structure II (sII) tetrahydrofuran (THF) hydrate crystal morphologies by adhering to the hydrate surface and inhibiting growth in a similar fashion to the kinetic inhibitor poly-N-vinylpyrrolidone (PVP). The effects of AFPs on the formation and growth rate of high-pressure sII gas mix hydrate demonstrated that AFPs are superior hydrate inhibitors compared to PVP. These results indicate that AFPs may be suitable for the study of new inhibitor systems and represent an important step towards the development of biologically-based hydrate inhibitors. PMID:20161789

  3. Antifreeze Proteins Modify the Freezing Process In Planta12

    PubMed Central

    Griffith, Marilyn; Lumb, Chelsey; Wiseman, Steven B.; Wisniewski, Michael; Johnson, Robert W.; Marangoni, Alejandro G.

    2005-01-01

    During cold acclimation, winter rye (Secale cereale L. cv Musketeer) plants accumulate antifreeze proteins (AFPs) in the apoplast of leaves and crowns. The goal of this study was to determine whether these AFPs influence survival at subzero temperatures by modifying the freezing process or by acting as cryoprotectants. In order to inhibit the growth of ice, AFPs must be mobile so that they can bind to specific sites on the ice crystal lattice. Guttate obtained from cold-acclimated winter rye leaves exhibited antifreeze activity, indicating that the AFPs are free in solution. Infrared video thermography was used to observe freezing in winter rye leaves. In the absence of an ice nucleator, AFPs had no effect on the supercooling temperature of the leaves. However, in the presence of an ice nucleator, AFPs lowered the temperature at which the leaves froze by 0.3°C to 1.2°C. In vitro studies showed that apoplastic proteins extracted from cold-acclimated winter rye leaves inhibited the recrystallization of ice and also slowed the rate of migration of ice through solution-saturated filter paper. When we examined the possible role of winter rye AFPs in cryoprotection, we found that lactate dehydrogenase activity was higher after freezing in the presence of AFPs compared with buffer, but the same effect was obtained by adding bovine serum albumin. AFPs had no effect on unstacked thylakoid volume after freezing, but did inhibit stacking of the thylakoids, thus indicating a loss of thylakoid function. We conclude that rye AFPs have no specific cryoprotective activity; rather, they interact directly with ice in planta and reduce freezing injury by slowing the growth and recrystallization of ice. PMID:15805474

  4. Extraction and Isolation of Antifreeze Proteins from Winter Rye (Secale cereale L.) Leaves.

    PubMed Central

    Hon, W. C.; Griffith, M.; Chong, P.; Yang, DSC.

    1994-01-01

    Apoplastic extracts of cold-acclimated winter rye (Secale cereale L. cv Musketeer) leaves were previously shown to exhibit antifreeze activity. The objectives of the present study were to identify and characterize individual antifreeze proteins present in the apoplastic extracts. The highest protein concentrations and antifreeze activity were obtained when the leaf apoplast was extracted with ascorbic acid and either CaCl2 or MgSO4. Seven major polypeptides were purified from these extracts by one-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis under nonreducing conditions. The five larger polypeptides, of 19, 26, 32, 34, and 36 kD, exhibited significant levels of antifreeze activity, whereas the 11- and 13-kD polypeptides showed only weak activity. Five of these polypeptides migrated with higher apparent molecular masses on SDS gels after treatment with 0.1 M dithiothreitol, which indicated the presence of intramolecular disulfide bonds. The apparent reduction of the disulfide bonds did not eliminate antifreeze activity in four of the polypeptides that contained intramolecular disulfide bonds and exhibited significant levels of antifreeze activity. The amino acid compositions of these polypeptides were similar in that they were all relatively enriched in the residues Asp/Asn, Glu/Gln, Ser, Thr, Gly, and Ala; they all lacked His, except for the 26-kD polypeptide, and they contained up to 5% Cys residues. These polypeptides were examined with antisera to other cystine-containing antifreeze proteins from fish and insects, and no common epitopes were detected. We conclude that cold-acclimated winter rye leaves produce multiple polypeptides with antifreeze activity that appear to be distinct from antifreezes produced by fish and insects. PMID:12232141

  5. Solvation structure of ice-binding antifreeze proteins

    NASA Astrophysics Data System (ADS)

    Hansen-Goos, Hendrik; Wettlaufer, John

    2009-03-01

    Antifreeze proteins (AFPs) can be found in organisms which survive at subzero temperatures. They were first discovered in polar fishes since the 1950's [1] and have been isolated meanwhile also from insects, plants, and bacteria. While AFPs shift the freezing point of water below the bulk melting point and hence can prevent recrystallization; the effect is non-colligative and there is a pronounced hysteresis between freezing and melting. For many AFPs it is generally accepted that they function through an irreversible binding to the ice-water interface which leads to a piecewise convex growth front with a lower nonequilibrium freezing point due to the Kelvin effect. Recent molecular dynamics simulations of the AFP from Choristoneura fumiferana reveal that the solvation structures of water at ice-binding and non-ice-binding faces of the protein are crucial for understanding how the AFP binds to the ice surface and how it is protected from being overgrown [2]. We use density functional theory of classical fluids in order to assess the microscopic solvent structure in the vicinity of protein faces with different surface properties. With our method, binding energies of different protein faces to the water-ice-interface can be computed efficiently in a simplified model. [1] Y. Yeh and R.E. Feeney, Chem. Rev. 96, 601 (1996). [2] D.R. Nutt and J.C. Smith, J. Am. Chem. Soc. 130, 13066 (2008).

  6. Long-range protein-water dynamics in hyperactive insect antifreeze proteins.

    PubMed

    Meister, Konrad; Ebbinghaus, Simon; Xu, Yao; Duman, John G; DeVries, Arthur; Gruebele, Martin; Leitner, David M; Havenith, Martina

    2013-01-29

    Antifreeze proteins (AFPs) are specific proteins that are able to lower the freezing point of aqueous solutions relative to the melting point. Hyperactive AFPs, identified in insects, have an especially high ability to depress the freezing point by far exceeding the abilities of other AFPs. In previous studies, we postulated that the activity of AFPs can be attributed to two distinct molecular mechanisms: (i) short-range direct interaction of the protein surface with the growing ice face and (ii) long-range interaction by protein-induced water dynamics extending up to 20 Å from the protein surface. In the present paper, we combine terahertz spectroscopy and molecular simulations to prove that long-range protein-water interactions make essential contributions to the high antifreeze activity of insect AFPs from the beetle Dendroides canadensis. We also support our hypothesis by studying the effect of the addition of the osmolyte sodium citrate.

  7. Dynamical mechanism of antifreeze proteins to prevent ice growth

    NASA Astrophysics Data System (ADS)

    Kutschan, B.; Morawetz, K.; Thoms, S.

    2014-08-01

    The fascinating ability of algae, insects, and fishes to survive at temperatures below normal freezing is realized by antifreeze proteins (AFPs). These are surface-active molecules and interact with the diffusive water-ice interface thus preventing complete solidification. We propose a dynamical mechanism on how these proteins inhibit the freezing of water. We apply a Ginzburg-Landau-type approach to describe the phase separation in the two-component system (ice, AFP). The free-energy density involves two fields: one for the ice phase with a low AFP concentration and one for liquid water with a high AFP concentration. The time evolution of the ice reveals microstructures resulting from phase separation in the presence of AFPs. We observed a faster clustering of pre-ice structure connected to a locking of grain size by the action of AFP, which is an essentially dynamical process. The adsorption of additional water molecules is inhibited and the further growth of ice grains stopped. The interfacial energy between ice and water is lowered allowing the AFPs to form smaller critical ice nuclei. Similar to a hysteresis in magnetic materials we observe a thermodynamic hysteresis leading to a nonlinear density dependence of the freezing point depression in agreement with the experiments.

  8. Transcription of antifreeze protein genes in Choristoneura fumiferana.

    PubMed

    Qin, W; Doucet, D; Tyshenko, M G; Walker, V K

    2007-08-01

    Antifreeze proteins (AFPs) are encoded by approximately 17 genes in the spruce budworm, Choristoneura fumiferana. Northern analysis using 6 different cDNA probes showed isoform-specific patterns that varied during development. Transcripts for the majority of isoforms were most abundant in the second instar overwintering stage, but some were also detected in first instar and even in egg stages. In situ hybridization using riboprobes corresponding to two 9 kDa protein isoforms showed differential AFP expression even in second instars; CfAFP10 RNA was detected in all tissues, but CfAFP337 RNA distribution was more limited. Two genomic regions encoding three AFP genes have been isolated. Presumptive regulatory regions conferred transcriptional activity when placed upstream of a luciferase reporter sequence and transfected into a C. fumiferana cell line. The CfAFP2.26 core promoter is an 87 bp sequence containing a TATA box, whereas the CfAFP2.7 core promoter is a 76 bp sequence with both a TATA box and CAAT box, which directed higher reporter activities when tested in vitro. Reporter activity was not enhanced with five different hormones, although lower activities were observed with all intron-containing constructs. AFP message half-life, as assessed using reporter assays, was not appreciably influenced by isoform-specific-3'UTRs. These studies successfully demonstrate the temporal and spatial diversity of AFP expression encoded by this small gene family, and underscore the complexity of their regulation.

  9. Dynamical mechanism of antifreeze proteins to prevent ice growth.

    PubMed

    Kutschan, B; Morawetz, K; Thoms, S

    2014-08-01

    The fascinating ability of algae, insects, and fishes to survive at temperatures below normal freezing is realized by antifreeze proteins (AFPs). These are surface-active molecules and interact with the diffusive water-ice interface thus preventing complete solidification. We propose a dynamical mechanism on how these proteins inhibit the freezing of water. We apply a Ginzburg-Landau-type approach to describe the phase separation in the two-component system (ice, AFP). The free-energy density involves two fields: one for the ice phase with a low AFP concentration and one for liquid water with a high AFP concentration. The time evolution of the ice reveals microstructures resulting from phase separation in the presence of AFPs. We observed a faster clustering of pre-ice structure connected to a locking of grain size by the action of AFP, which is an essentially dynamical process. The adsorption of additional water molecules is inhibited and the further growth of ice grains stopped. The interfacial energy between ice and water is lowered allowing the AFPs to form smaller critical ice nuclei. Similar to a hysteresis in magnetic materials we observe a thermodynamic hysteresis leading to a nonlinear density dependence of the freezing point depression in agreement with the experiments.

  10. Antifreeze proteins in winter rye are similar to pathogenesis-related proteins.

    PubMed Central

    Hon, W C; Griffith, M; Mlynarz, A; Kwok, Y C; Yang, D S

    1995-01-01

    The ability to control extracellular ice formation during freezing is critical to the survival of freezing-tolerant plants. Antifreeze proteins, which are proteins that have the ability to retard ice crystal growth, were recently identified as the most abundant apoplastic proteins in cold-acclimated winter rye (Secale cereale L.) leaves. In the experiments reported here, amino-terminal sequence comparisons, immuno-cross-reactions, and enzyme activity assays all indicated that these antifreeze proteins are similar to members of three classes of pathogenesis-related proteins, namely, endochitinases, endo-beta-1,3-glucanases, and thaumatin-like proteins. Apoplastic endochitinases and endo-beta-1,3-glucanases that were induced by pathogens in freezing-sensitive tobacco did not exhibit antifreeze activity. Our findings suggest that subtle structural differences may have evolved in the pathogenesis-related proteins that accumulate at cold temperatures in winter rye to confer upon these proteins the ability to bind to ice. PMID:8552719

  11. Anchored Clathrate Waters Bind Antifreeze Proteins to Ice

    SciTech Connect

    C Garnham; R Campbell; P Davies

    2011-12-31

    The mechanism by which antifreeze proteins (AFPs) irreversibly bind to ice has not yet been resolved. The ice-binding site of an AFP is relatively hydrophobic, but also contains many potential hydrogen bond donors/acceptors. The extent to which hydrogen bonding and the hydrophobic effect contribute to ice binding has been debated for over 30 years. Here we have elucidated the ice-binding mechanism through solving the first crystal structure of an Antarctic bacterial AFP. This 34-kDa domain, the largest AFP structure determined to date, folds as a Ca{sup 2+}-bound parallel beta-helix with an extensive array of ice-like surface waters that are anchored via hydrogen bonds directly to the polypeptide backbone and adjacent side chains. These bound waters make an excellent three-dimensional match to both the primary prism and basal planes of ice and in effect provide an extensive X-ray crystallographic picture of the AFP{vert_ellipsis}ice interaction. This unobstructed view, free from crystal-packing artefacts, shows the contributions of both the hydrophobic effect and hydrogen bonding during AFP adsorption to ice. We term this mode of binding the 'anchored clathrate' mechanism of AFP action.

  12. Tenebrio molitor antifreeze protein gene identification and regulation.

    PubMed

    Qin, Wensheng; Walker, Virginia K

    2006-02-15

    The yellow mealworm, Tenebrio molitor, is a freeze susceptible, stored product pest. Its winter survival is facilitated by the accumulation of antifreeze proteins (AFPs), encoded by a small gene family. We have now isolated 11 different AFP genomic clones from 3 genomic libraries. All the clones had a single coding sequence, with no evidence of intervening sequences. Three genomic clones were further characterized. All have putative TATA box sequences upstream of the coding regions and multiple potential poly(A) signal sequences downstream of the coding regions. A TmAFP regulatory region, B1037, conferred transcriptional activity when ligated to a luciferase reporter sequence and after transfection into an insect cell line. A 143 bp core promoter including a TATA box sequence was identified. Its promoter activity was increased 4.4 times by inserting an exotic 245 bp intron into the construct, similar to the enhancement of transgenic expression seen in several other systems. The addition of a duplication of the first 120 bp sequence from the 143 bp core promoter decreased promoter activity by half. Although putative hormonal response sequences were identified, none of the five hormones tested enhanced reporter activity. These studies on the mechanisms of AFP transcriptional control are important for the consideration of any transfer of freeze-resistance phenotypes to beneficial hosts.

  13. Anchored clathrate waters bind antifreeze proteins to ice.

    PubMed

    Garnham, Christopher P; Campbell, Robert L; Davies, Peter L

    2011-05-03

    The mechanism by which antifreeze proteins (AFPs) irreversibly bind to ice has not yet been resolved. The ice-binding site of an AFP is relatively hydrophobic, but also contains many potential hydrogen bond donors/acceptors. The extent to which hydrogen bonding and the hydrophobic effect contribute to ice binding has been debated for over 30 years. Here we have elucidated the ice-binding mechanism through solving the first crystal structure of an Antarctic bacterial AFP. This 34-kDa domain, the largest AFP structure determined to date, folds as a Ca(2+)-bound parallel beta-helix with an extensive array of ice-like surface waters that are anchored via hydrogen bonds directly to the polypeptide backbone and adjacent side chains. These bound waters make an excellent three-dimensional match to both the primary prism and basal planes of ice and in effect provide an extensive X-ray crystallographic picture of the AFPice interaction. This unobstructed view, free from crystal-packing artefacts, shows the contributions of both the hydrophobic effect and hydrogen bonding during AFP adsorption to ice. We term this mode of binding the "anchored clathrate" mechanism of AFP action.

  14. Ice-binding structure and mechanism of an antifreeze protein from winter flounder.

    PubMed

    Sicheri, F; Yang, D S

    1995-06-01

    Antifreeze proteins provide fish with protection against the freezing effect of polar environments by binding to ice surfaces and inhibiting growth of ice crystals. We present the X-ray crystal structure at 1.5 A resolution of a lone alpha-helical antifreeze protein from winter flounder, which provides a detailed look at its ice-binding features. These consist of four repeated ice-binding motifs, the side chains of which are inherently rigid or restrained by pair-wise side-chain interactions to form a flat binding surface. Elaborate amino- and carboxy-terminal cap structures are also present, which explain the protein's rich alpha-helical content in solution. We propose an ice-binding model that accounts for the binding specificity of the antifreeze protein along the <0112> axes of the (2021) ice planes.

  15. Cloning and expression of Tenebrio molitor antifreeze protein in Escherichia coli.

    PubMed

    Yue, Chang-Wu; Zhang, Yi-Zheng

    2009-03-01

    A novel antifreeze protein cDNA was cloned by RT-PCR from the larva of the yellow mealworm Tenebrio molitor. The coding fragment of 339 bp encodes a protein of 112 amino acid residues and was fused to the expression vectors pET32a and pTWIN1. The resulted expression plasmids were transformed into Escherischia coli strains BL21 (DE3), ER2566, and Origami B (DE3), respectively. Several strategies were used for expression of the highly disulfide-bonded beta-helix-contained protein with the activity of antifreeze in different expression systems. A protocol for production of refolded and active T. molitor antifreeze protein in bacteria was obtained.

  16. Stable, high-level expression of a type I antifreeze protein in Escherichia coli.

    PubMed

    Solomon, R G; Appels, R

    1999-06-01

    The type I antifreeze proteins are simple amphipathic helical proteins found in abundance in polar fish species, where they act to prevent freezing of internal fluids by a mechanism of noncolligative freezing point depression. Large-scale production of these proteins for research and biotechnological purposes has been hampered by their apparent instability when expressed in heterologous host systems. This has necessitated their production as fusion proteins, in polymeric form, or as proproteins for secretion, with the concomitant necessity for postpurification processing to generate the mature form of the protein. We have successfully expressed a recombinant variant of type I antifreeze protein (rAFP) in Escherichia coli using the inducible T7 polymerase transcription expression system. The rAFP contains five copies of the 11 amino acid ice-binding repeat motif found in all type I antifreeze proteins. The protein accumulates to high levels intracellularly in the form of inclusion bodies, with no apparent degradation by the cellular proteolytic machinery. We have devised a simple and rapid purification protocol for this recombinant type I antifreeze protein which does not require cellular fractionation, purification of the inclusion bodies, or chromatographic steps. This protocol may be of general use for this class of protein. The protein displays all three activities common to these proteins: recrystallization inhibition, noncolligative freezing point depression, and modification of the morphology of single ice crystals in solution.

  17. Blocking rapid ice crystal growth through nonbasal plane adsorption of antifreeze proteins

    PubMed Central

    Olijve, Luuk L. C.; Meister, Konrad; DeVries, Arthur L.; Duman, John G.; Guo, Shuaiqi; Bakker, Huib J.; Voets, Ilja K.

    2016-01-01

    Antifreeze proteins (AFPs) are a unique class of proteins that bind to growing ice crystal surfaces and arrest further ice growth. AFPs have gained a large interest for their use in antifreeze formulations for water-based materials, such as foods, waterborne paints, and organ transplants. Instead of commonly used colligative antifreezes such as salts and alcohols, the advantage of using AFPs as an additive is that they do not alter the physicochemical properties of the water-based material. Here, we report the first comprehensive evaluation of thermal hysteresis (TH) and ice recrystallization inhibition (IRI) activity of all major classes of AFPs using cryoscopy, sonocrystallization, and recrystallization assays. The results show that TH activities determined by cryoscopy and sonocrystallization differ markedly, and that TH and IRI activities are not correlated. The absence of a distinct correlation in antifreeze activity points to a mechanistic difference in ice growth inhibition by the different classes of AFPs: blocking fast ice growth requires rapid nonbasal plane adsorption, whereas basal plane adsorption is only relevant at long annealing times and at small undercooling. These findings clearly demonstrate that biomimetic analogs of antifreeze (glyco)proteins should be tailored to the specific requirements of the targeted application. PMID:26936953

  18. Blocking rapid ice crystal growth through nonbasal plane adsorption of antifreeze proteins.

    PubMed

    Olijve, Luuk L C; Meister, Konrad; DeVries, Arthur L; Duman, John G; Guo, Shuaiqi; Bakker, Huib J; Voets, Ilja K

    2016-04-05

    Antifreeze proteins (AFPs) are a unique class of proteins that bind to growing ice crystal surfaces and arrest further ice growth. AFPs have gained a large interest for their use in antifreeze formulations for water-based materials, such as foods, waterborne paints, and organ transplants. Instead of commonly used colligative antifreezes such as salts and alcohols, the advantage of using AFPs as an additive is that they do not alter the physicochemical properties of the water-based material. Here, we report the first comprehensive evaluation of thermal hysteresis (TH) and ice recrystallization inhibition (IRI) activity of all major classes of AFPs using cryoscopy, sonocrystallization, and recrystallization assays. The results show that TH activities determined by cryoscopy and sonocrystallization differ markedly, and that TH and IRI activities are not correlated. The absence of a distinct correlation in antifreeze activity points to a mechanistic difference in ice growth inhibition by the different classes of AFPs: blocking fast ice growth requires rapid nonbasal plane adsorption, whereas basal plane adsorption is only relevant at long annealing times and at small undercooling. These findings clearly demonstrate that biomimetic analogs of antifreeze (glyco)proteins should be tailored to the specific requirements of the targeted application.

  19. Immunolocalization of Antifreeze Proteins in Winter Rye Leaves, Crowns, and Roots by Tissue Printing.

    PubMed Central

    Antikainen, M.; Griffith, M.; Zhang, J.; Hon, W. C.; Yang, DSC.; Pihakaski-Maunsbach, K.

    1996-01-01

    During cold acclimation, antifreeze proteins (AFPs) that are similar to pathogenesis-related proteins accumulate in the apoplast of winter rye (Secale cereale L. cv Musketeer) leaves. AFPs have the ability to modify the growth of ice. To elucidate the role of AFPs in the freezing process, they were assayed and immunolocalized in winter rye leaves, crowns, and roots. Each of the total soluble protein extracts from cold-acclimated rye leaves, crowns, and roots exhibited antifreeze activity, whereas no antifreeze activity was observed in extracts from nonacclimated rye plants. Antibodies raised against three apoplastic rye AFPs, corresponding to a glucanase-like protein (GLP, 32 kD), a chitinase-like protein (CLP, 35 kD), and a thaumatin-like protein (TLP, 25 kD), were used in tissue printing to show that the AFPs are localized in the epidermis and in cells surrounding intercellular spaces in cold-acclimated plants. Although GLPs, CLPs, and TLPs were present in nonacclimated plants, they were found in different locations and did not exhibit antifreeze activity, which suggests that different isoforms of pathogenesis-related proteins are produced at low temperature. The location of rye AFPs may prevent secondary nucleation of cells by epiphytic ice or by ice propagating through the xylem. The distributions of pathogenesis-induced and cold-accumulated GLPs, CLPs, and TLPs are similar and may reflect the common pathways by which both pathogens and ice enter and propagate through plant tissues. PMID:12226223

  20. Unusual structural properties of water within the hydration shell of hyperactive antifreeze protein

    NASA Astrophysics Data System (ADS)

    Kuffel, Anna; Czapiewski, Dariusz; Zielkiewicz, Jan

    2014-08-01

    Many hypotheses can be encountered explaining the mechanism of action of antifreeze proteins. One widespread theory postulates that the similarity of structural properties of solvation water of antifreeze proteins to ice is crucial to the antifreeze activity of these agents. In order to investigate this problem, the structural properties of solvation water of the hyperactive antifreeze protein from Choristoneura fumiferana were analyzed and compared with the properties of solvation water present at the surface of ice. The most striking observations concerned the temperature dependence of changes in water structure. In the case of solvation water of the ice-binding plane, the difference between the overall structural ordering of solvation water and bulk water diminished with increasing temperature; in the case of solvation water of the rest of the protein, the trend was opposite. In this respect, the solvation water of the ice-binding plane roughly resembled the hydration layer of ice. Simultaneously, the whole solvation shell of the protein displayed some features that are typical for solvation shells of many other proteins and are not encountered in the solvation water of ice. In the first place, this is an increase in density of water around the protein. The opposite is true for the solvation water of ice - it is less dense than bulk water. Therefore, even though the structure of solvation water of ice-binding plane and the structure of solvation water of ice seem to share some similarities, densitywise they differ.

  1. Purification, cDNA cloning and recombinant protein expression of a phloem lectin-like anti-insect defense protein BPLP from the phloem exudate of the wax gourd, Benincasa hispida.

    PubMed

    Ota, Eiji; Tsuchiya, Wataru; Yamazaki, Toshimasa; Nakamura, Masatoshi; Hirayama, Chikara; Konno, Kotaro

    2013-05-01

    Latex and other exudates in plants contain various proteins that are thought to play important defensive roles against herbivorous insects and pathogens. Herein, the defensive effects of phloem exudates against the Eri silkworm, Samia ricini (Saturniidae, Lepidoptera) in several cucurbitaceous plants were investigated. It was found that phloem exudates are responsible for the defensive activities of cucurbitaceous plants, such as the wax gourd Benincasa hispida and Cucumis melo, especially in B. hispida, whose leaves showed the strongest growth-inhibitory activity of all the cucurbitaceous plants tested. A 35 kDa proteinaceous growth-inhibitory factor against insects designated BPLP (B. hispida Phloem Lectin-like Protein) was next isolated and purified from the B. hispida exudate, using anion exchange and gel filtration chromatography. A very low concentration (70 μg/g) of BPLP significantly inhibited growth of S. ricini larvae. The full-length cDNA (1076 bp) encoding BPLP was cloned and its nucleotide sequence was determined. The deduced amino acid sequence of BPLP had 51% identity with a cucurbitaceous phloem lectin (phloem protein 2, PP2), and showed binding specificity to oligomers of N-acetylglucosamine. Some features of BPLP indicated that it does not have a cysteine residue and it is composed of two repeats of similar sequences, suggesting that BPLP is distinct from PP2. Recombinant BPLP, obtained by expressing the cDNA in Escherichia coli, showed both chitin-binding lectin activity and growth-inhibitory activity against S. ricini larvae. The present study thus provides experimental evidence that phloem exudates of Cucurbitaceae plants, analogous to plant latex, play defensive roles against insect herbivores, especially against chewing insects, and contain defensive substances toxic to them.

  2. Lectin-like molecules in transcriptome of Littorina littorea hemocytes.

    PubMed

    Gorbushin, Alexander M; Borisova, Elena A

    2015-01-01

    The common periwinkle Littorina littorea was introduced in the list of models for comparative immunobiology as a representative of phylogenetically important taxon Caenogastropoda. Using Illumina sequencing technology, we de novo assembled the transcriptome of Littorina littorea hemocytes from 182 million mRNA-Seq pair-end 100 bp reads into a total of 15,526 contigs clustered in 4472 unigenes. The transcriptome profile was analyzed for presence of carbohydrate-binding molecules in a variety of architectural contexts. Hemocytes' repertoire of lectin-like proteins bearing conserved carbohydrate-recognition domains (CRDs) is highly diversified, including 11 of 15 lectin families earlier described in animals, as well as the novel members of lectin family found for the first time in mollusc species. The new molluscan lineage-specific domain combinations were confirmed by cloning and sequencing, including the fuco-lectin related molecules (FLReMs) composed of N-terminal region with no sequence homology to any known protein, a middle Fucolectin Tachylectin-4 Pentaxrin (FTP) domain, and a C-terminal epidermal growth factor (EGF) repeat region. The repertoire of lectin-like molecules is discussed in terms of their potential participation in the receptor phase of immune response. In total, immune-associated functions may be attributed to 70 transcripts belonging to 6 lectin families. These lectin-like genes show low overlap between species of invertebrates, suggesting relatively rapid evolution of immune-associated genes in the group. The repertoire provides valuable candidates for further characterization of the gene functions in mollusc immunity.

  3. Long-range protein–water dynamics in hyperactive insect antifreeze proteins

    PubMed Central

    Meister, Konrad; Ebbinghaus, Simon; Xu, Yao; Duman, John G.; DeVries, Arthur; Gruebele, Martin; Leitner, David M.; Havenith, Martina

    2013-01-01

    Antifreeze proteins (AFPs) are specific proteins that are able to lower the freezing point of aqueous solutions relative to the melting point. Hyperactive AFPs, identified in insects, have an especially high ability to depress the freezing point by far exceeding the abilities of other AFPs. In previous studies, we postulated that the activity of AFPs can be attributed to two distinct molecular mechanisms: (i) short-range direct interaction of the protein surface with the growing ice face and (ii) long-range interaction by protein-induced water dynamics extending up to 20 Å from the protein surface. In the present paper, we combine terahertz spectroscopy and molecular simulations to prove that long-range protein–water interactions make essential contributions to the high antifreeze activity of insect AFPs from the beetle Dendroides canadensis. We also support our hypothesis by studying the effect of the addition of the osmolyte sodium citrate. PMID:23277543

  4. Function of the hydration layer around an antifreeze protein revealed by atomistic molecular dynamics simulations

    SciTech Connect

    Nutt, David; Smith, Jeremy C

    2008-10-01

    Atomistic molecular dynamics simulations are used to investigate the mechanism by which the antifreeze protein from the spruce budworm, Choristoneura fumiferana, binds to ice. Comparison of structural and dynamic properties of the water around the three faces of the triangular prism-shaped protein in aqueous solution reveals that at low temperature the water structure is ordered and the dynamics slowed down around the ice-binding face of the protein, with a disordering effect observed around the other two faces. These results suggest a dual role for the solvation water around the protein. The preconfigured solvation shell around the ice-binding face is involved in the initial recognition and binding of the antifreeze protein to ice by lowering the barrier for binding and consolidation of the protein:ice interaction surface. Thus, the antifreeze protein can bind to the molecularly rough ice surface by becoming actively involved in the formation of its own binding site. Also, the disruption of water structure around the rest of the protein helps prevent the adsorbed protein becoming covered by further ice growth.

  5. Applications of type I antifreeze proteins: studies with model membranes & cryoprotectant properties.

    PubMed

    Inglis, Steven R; Turner, Jennifer J; Harding, Margaret M

    2006-12-01

    Antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs), found in the body fluids of many species of polar fish allow them to survive in waters colder than the equilibrium freezing point of their blood and other internal fluids. Despite their structural diversity, all AF(G)Ps kinetically depress the temperature at which ice grows in a non-colligative manner and hence exhibit thermal hysteresis. AF(G)Ps also share the ability to interact with and protect mammalian cells and tissues from hypothermic damage (e.g., improved storage of human blood platelets at low temperatures), and are able to stabilize or disrupt membrane composition during low temperature and freezing stress (e.g., cryoprotectant properties in stabilization of sperm and oocytes). This review will summarize studies of AFPs with phospholipids and plant lipids, proposed mechanisms for inhibition of leakage from membranes, and cryoprotectant studies with biological samples. The major focus will be on the alpha-helical type I antifreeze proteins, and synthetic mutants, that have been most widely studied. For completeness, data on glycoproteins will also be presented. While a number of models to explain stabilization and destabilization of different lipid systems have been proposed, it is currently not possible to predict whether a particular AFP will stabilize or destabilize a given lipid system. Furthermore the relationship between the antifreeze property of thermal hysteresis and membrane stabilization is unknown. This lack of detailed knowledge about how AFPs function in the presence of different types of materials has hampered progress toward the development of antifreezes for cold storage of cells, tissues, and organs.

  6. Marine Antifreeze Proteins: Structure, Function, and Application to Cryopreservation as a Potential Cryoprotectant

    PubMed Central

    Kim, Hak Jun; Lee, Jun Hyuck; Hur, Young Baek; Lee, Chang Woo; Park, Sun-Ha; Koo, Bon-Won

    2017-01-01

    Antifreeze proteins (AFPs) are biological antifreezes with unique properties, including thermal hysteresis (TH), ice recrystallization inhibition (IRI), and interaction with membranes and/or membrane proteins. These properties have been utilized in the preservation of biological samples at low temperatures. Here, we review the structure and function of marine-derived AFPs, including moderately active fish AFPs and hyperactive polar AFPs. We also survey previous and current reports of cryopreservation using AFPs. Cryopreserved biological samples are relatively diverse ranging from diatoms and reproductive cells to embryos and organs. Cryopreserved biological samples mainly originate from mammals. Most cryopreservation trials using marine-derived AFPs have demonstrated that addition of AFPs can improve post-thaw viability regardless of freezing method (slow-freezing or vitrification), storage temperature, and types of biological sample type. PMID:28134801

  7. Ice shaping properties, similar to that of antifreeze proteins, of a zirconium acetate complex.

    PubMed

    Deville, Sylvain; Viazzi, Céline; Leloup, Jérôme; Lasalle, Audrey; Guizard, Christian; Maire, Eric; Adrien, Jérôme; Gremillard, Laurent

    2011-01-01

    The control of the growth morphologies of ice crystals is a critical issue in fields as diverse as biomineralization, medicine, biology, civil or food engineering. Such control can be achieved through the ice-shaping properties of specific compounds. The development of synthetic ice-shaping compounds is inspired by the natural occurrence of such properties exhibited by antifreeze proteins. We reveal how a particular zirconium acetate complex is exhibiting ice-shaping properties very similar to that of antifreeze proteins, albeit being a radically different compound. We use these properties as a bioinspired approach to template unique faceted pores in cellular materials. These results suggest that ice-structuring properties are not exclusive to long organic molecules and should broaden the field of investigations and applications of such substances.

  8. Ice Shaping Properties, Similar to That of Antifreeze Proteins, of a Zirconium Acetate Complex

    PubMed Central

    Deville, Sylvain; Viazzi, Céline; Leloup, Jérôme; Lasalle, Audrey; Guizard, Christian; Maire, Eric; Adrien, Jérôme; Gremillard, Laurent

    2011-01-01

    The control of the growth morphologies of ice crystals is a critical issue in fields as diverse as biomineralization, medicine, biology, civil or food engineering. Such control can be achieved through the ice-shaping properties of specific compounds. The development of synthetic ice-shaping compounds is inspired by the natural occurrence of such properties exhibited by antifreeze proteins. We reveal how a particular zirconium acetate complex is exhibiting ice-shaping properties very similar to that of antifreeze proteins, albeit being a radically different compound. We use these properties as a bioinspired approach to template unique faceted pores in cellular materials. These results suggest that ice-structuring properties are not exclusive to long organic molecules and should broaden the field of investigations and applications of such substances. PMID:22028886

  9. A two-dimensional adsorption kinetic model for thermal hysteresis activity in antifreeze proteins

    NASA Astrophysics Data System (ADS)

    Li, Q. Z.; Yeh, Y.; Liu, J. J.; Feeney, R. E.; Krishnan, V. V.

    2006-05-01

    Antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs), collectively abbreviated as AF(G)Ps, are synthesized by various organisms to enable their cells to survive in subzero environments. Although the AF(G)Ps are markedly diverse in structure, they all function by adsorbing to the surface of embryonic ice crystals to inhibit their growth. This adsorption results in a freezing temperature depression without an appreciable change in the melting temperature. The difference between the melting and freezing temperatures, termed thermal hysteresis (TH), is used to detect and quantify the antifreeze activity. Insights from crystallographic structures of a number of AFPs have led to a good understanding of the ice-protein interaction features. Computational studies have focused either on verifying a specific model of AFP-ice interaction or on understanding the protein-induced changes in the ice crystal morphology. In order to explain the origin of TH, we propose a novel two-dimensional adsorption kinetic model between AFPs and ice crystal surfaces. The validity of the model has been demonstrated by reproducing the TH curve on two different β-helical AFPs upon increasing the protein concentration. In particular, this model is able to accommodate the change in the TH behavior observed experimentally when the size of the AFPs is increased systematically. Our results suggest that in addition to the specificity of the AFPs for the ice, the coverage of the AFPs on the ice surface is an equally necessary condition for their TH activity.

  10. Adsorption thermodynamics of two-domain antifreeze proteins: theory and Monte Carlo simulations.

    PubMed

    Narambuena, Claudio F; Sanchez Varretti, Fabricio O; Ramirez-Pastor, Antonio J

    2016-09-21

    In this paper we develop the statistical thermodynamics of two-domain antifreeze proteins adsorbed on ice. We use a coarse-grained model and a lattice network in order to represent the protein and ice, respectively. The theory is obtained by combining the exact analytical expression for the partition function of non-interacting linear k-mers adsorbed in one dimension, and its extension to higher dimensions. The total and partial adsorption isotherms, and the coverage and temperature dependence of the Helmholtz free energy and configurational entropy are given. The formalism reproduces the classical Langmuir equation, leads to the exact statistical thermodynamics of molecules adsorbed in one dimension, and provides a close approximation for two-dimensional systems. Comparisons with analytical data obtained using the modified Langmuir model (MLM) and Monte Carlo simulations in the grand canonical ensemble were performed in order to test the validity of the theoretical predictions. In the MC calculations, the different mechanisms proposed in the literature to describe the adsorption of two-domain antifreeze proteins on ice were analyzed. Indistinguishable results were obtained in all cases, which verifies the thermodynamic equivalence of these mechanisms and allows the choice of the most suitable mechanism for theoretical studies of equilibrium properties. Even though a good qualitative agreement is obtained between MLM and MC data, it is found that the new theoretical framework offers a more accurate description of the phenomenon of adsorption of two-domain antifreeze proteins.

  11. Rational, yet simple, design and synthesis of an antifreeze-protein inspired polymer for cellular cryopreservation.

    PubMed

    Mitchell, Daniel E; Cameron, Neil R; Gibson, Matthew I

    2015-08-21

    Antifreeze (glyco) proteins AF(G)Ps are potent ice recrystallization inhibitors, which is a desirable property to enhance cryopreservation of donor tissue/cells. Here we present the rational synthesis of a new, biomimetic, ice-recrystallization inhibiting polymer derived from a cheap commodity polymer, based on an ampholyte structure. The polymer is used to enhance the cryopreservation of red blood cells, demonstrating a macromolecular solution to tissue storage.

  12. Partitioning of fish and insect antifreeze proteins into ice suggests they bind with comparable affinity.

    PubMed

    Marshall, Christopher B; Tomczak, Melanie M; Gauthier, Sherry Y; Kuiper, Michael J; Lankin, Christopher; Walker, Virginia K; Davies, Peter L

    2004-01-13

    Antifreeze proteins (AFPs) inhibit the growth of ice by binding to the surface of ice crystals, preventing the addition of water molecules to cause a local depression of the freezing point. AFPs from insects are much more effective at depressing the freezing point than fish AFPs. Here, we have investigated the possibility that insect AFPs bind more avidly to ice than fish AFPs. Because it is not possible to directly measure the affinity of an AFP for ice, we have assessed binding indirectly by examining the partitioning of proteins into a slowly growing ice hemisphere. AFP molecules adsorbed to the surface and became incorporated into the ice as they were overgrown. Solutes, including non-AFPs, were very efficiently excluded from ice, whereas AFPs became incorporated into ice at a concentration roughly equal to that of the original solution, and this was independent of the AFP concentration in the range (submillimolar) tested. Despite their >10-fold difference in antifreeze activity, fish and insect AFPs partitioned into ice to a similar degree, suggesting that insect AFPs do not bind to ice with appreciably higher affinity. Additionally, we have demonstrated that steric mutations on the ice binding surface that decrease the antifreeze activity of an AFP also reduce its inclusion into ice, supporting the validity of using partitioning measurements to assess a protein's affinity for ice.

  13. JACALIN-LECTIN LIKE1 Regulates the Nuclear Accumulation of GLYCINE-RICH RNA-BINDING PROTEIN7, Influencing the RNA Processing of FLOWERING LOCUS C Antisense Transcripts and Flowering Time in Arabidopsis1[OPEN

    PubMed Central

    Xiao, Jun; Li, Chunhua; Xu, Shujuan; Xing, Lijing; Xu, Yunyuan; Chong, Kang

    2015-01-01

    Lectins selectively recognize sugars or glycans for defense in living cells, but less is known about their roles in the development process and the functional network with other factors. Here, we show that Arabidopsis (Arabidopsis thaliana) JACALIN-LECTIN LIKE1 (AtJAC1) functions in flowering time control. Loss of function of AtJAC1 leads to precocious flowering, whereas overexpression of AtJAC1 causes delayed flowering. AtJAC1 influences flowering through regulation of the key flowering repressor gene FLOWERING LOCUS C (FLC). Genetic analysis revealed that AtJAC1’s function is mostly dependent on GLYCINE-RICH RNA-BINDING PROTEIN7 (GRP7), an upstream regulator of FLC. Biochemical and cell biological data indicated that AtJAC1 interacted physically with GRP7 specifically in the cytoplasm. AtJAC1 influences the nucleocytoplasmic distribution of GRP7, with predominant nuclear localization of GRP7 when AtJAC1 function is lost but retention of GRP7 in the cytoplasm when AtJAC1 is overexpressed. A temporal inducible assay suggested that AtJAC1’s regulation of flowering could be compromised by the nuclear accumulation of GRP7. In addition, GRP7 binds to the antisense precursor messenger RNA of FLC through a conserved RNA motif. Loss of GRP7 function leads to the elevation of total FLC antisense transcripts and reduced proximal-distal polyadenylation ratio, as well as histone methylation changes in the FLC gene body region and increased total functional sense FLC transcript. Attenuating the direct binding of GRP7 with competing artificial RNAs leads to changes of FLC antisense precursor messenger RNA processing and flowering transition. Taken together, our study indicates that AtJAC1 coordinates with GRP7 in shaping plant development through the regulation of RNA processing in Arabidopsis. PMID:26392261

  14. Intermolecular interaction studies of winter flounder antifreeze protein reveal the existence of thermally accessible binding state.

    PubMed

    Nguyen, Dat H; Colvin, Michael E; Yeh, Yin; Feeney, Robert E; Fink, William H

    2004-10-05

    The physical nature underlying intermolecular interactions between two rod-like winter flounder antifreeze protein (AFP) molecules and their implication for the mechanism of antifreeze function are examined in this work using molecular dynamics simulations, augmented with free energy calculations employing a continuum solvation model. The energetics for different modes of interactions of two AFP molecules is examined in both vacuum and aqueous phases along with the water distribution in the region encapsulated by two antiparallel AFP backbones. The results show that in a vacuum two AFP molecules intrinsically attract each other in the antiparallel fashion, where their complementary charge side chains face each other directly. In the aqueous environment, this attraction is counteracted by both screening and entropic effects. Therefore, two nearly energetically degenerate states, an aggregated state and a dissociated state, result as a new aspect of intermolecular interaction in the paradigm for the mechanism of action of AFP. The relevance of these findings to the mechanism of function of freezing inhibition in the context of our work on Antarctic cod antifreeze glycoprotein (Nguyen et al., Biophysical Journal, 2002, Vol. 82, pp. 2892-2905) is discussed.

  15. Convulxin, a C-type lectin-like protein, inhibits HCASMCs functions via WAD-motif/integrin-αv interaction and NF-κB-independent gene suppression of GRO and IL-8.

    PubMed

    Shih, Chun-Ho; Chiang, Tin-Bin; Wang, Wen-Jeng

    2017-03-15

    Convulxin (CVX), a C-type lectin-like protein (CLPs), is a potent platelet aggregation inducer. To evaluate its potential applications in angiogenic diseases, the multimeric CVX were further explored on its mode of actions toward human coronary artery smooth muscle cells (HCASMCs). The N-terminus of β-chain of CVX (CVX-β) contains a putative disintegrin-like domain with a conserved motif upon the sequence comparison with other CLPs. Importantly, native CVX had no cytotoxic activity as examined by electrophoretic pattern. A Trp-Ala-Asp (WAD)-containing octapeptide, MTWADAEK, was thereafter synthesized and analyzed in functional assays. In the case of specific integrin antagonists as positive controls, the anti-angiogenic effects of CVX on HCASMCs were investigated by series of functional analyses. CVX showed to exhibit multiple inhibitory activities toward HCASMCs proliferation, adhesion and invasion with a dose- and integrin αvβ3-dependent fashion. However, the WAD-octapeptide exerting a minor potency could also work as an active peptidomimetic. In addition, flow cytometric analysis demonstrated both the intact CVX and synthetic peptide can specifically interact with integrin-αv on HCASMCs and CVX was shown to have a down-regulatory effect on the gene expression of CXC-chemokines, such as growth-related oncogene and interleukin-8. According to nuclear factor-κB (NF-κB) p65 translocation assay and Western blotting analysis, the NF-κB activation was not involved in the signaling events of CVX-induced gene expression. In conclusion, CVX may act as a disintegrin-like protein via the interactions of WAD-motif in CVX-β with integrin-αv on HCASMCs and it also is a gene suppressor with the ability to diminish the expression of two CXC-chemokines in a NF-κB-independent manner. Indeed, more extensive investigations are needed and might create a new avenue for the development of a novel angiostatic agent.

  16. Unusual dynamic properties of water near the ice-binding plane of hyperactive antifreeze protein

    SciTech Connect

    Kuffel, Anna; Czapiewski, Dariusz; Zielkiewicz, Jan

    2015-10-07

    The dynamical properties of solvation water of hyperactive antifreeze protein from Choristoneura fumiferana (CfAFP) are analyzed and discussed in context of its antifreeze activity. The protein comprises of three well-defined planes and one of them binds to the surface of ice. The dynamical properties of solvation water around each of these planes were analyzed separately; the results are compared with the dynamical properties of solvation water of ice around its two crystallographic planes: basal and prism. Three main conclusions are inferred from our investigations. The first one is that the solvation shell of CfAFP does not seem to be particularly far-ranged, at least not beyond what is usually observed for proteins that do not interact with ice. Therefore, it does not appear to us that the antifreeze activity is enhanced by a long-ranged retardation of water mobility. Also the correlation between the collective mobility of water and the collective mobility of protein atoms highly resembles the one measured for the protein that does not interact with ice. Our second conclusion is that the dynamical properties of solvation water of CfAFP are non-uniform. The dynamics of solvation water of ice-binding plane is, in some respects, different from the dynamics of solvation water of the two remaining planes. The feature that distinguishes the dynamics of solvation water of the three planes is the activation energy of diffusion process. The third conclusion is that—from the three analyzed solvation shells of CfAFP—the dynamical properties of solvation water of the ice-binding plane resemble the most the properties of solvation water of ice; note, however, that these properties still clearly differ from the dynamic properties of solvation water of ice.

  17. Unusual dynamic properties of water near the ice-binding plane of hyperactive antifreeze protein

    NASA Astrophysics Data System (ADS)

    Kuffel, Anna; Czapiewski, Dariusz; Zielkiewicz, Jan

    2015-10-01

    The dynamical properties of solvation water of hyperactive antifreeze protein from Choristoneura fumiferana (CfAFP) are analyzed and discussed in context of its antifreeze activity. The protein comprises of three well-defined planes and one of them binds to the surface of ice. The dynamical properties of solvation water around each of these planes were analyzed separately; the results are compared with the dynamical properties of solvation water of ice around its two crystallographic planes: basal and prism. Three main conclusions are inferred from our investigations. The first one is that the solvation shell of CfAFP does not seem to be particularly far-ranged, at least not beyond what is usually observed for proteins that do not interact with ice. Therefore, it does not appear to us that the antifreeze activity is enhanced by a long-ranged retardation of water mobility. Also the correlation between the collective mobility of water and the collective mobility of protein atoms highly resembles the one measured for the protein that does not interact with ice. Our second conclusion is that the dynamical properties of solvation water of CfAFP are non-uniform. The dynamics of solvation water of ice-binding plane is, in some respects, different from the dynamics of solvation water of the two remaining planes. The feature that distinguishes the dynamics of solvation water of the three planes is the activation energy of diffusion process. The third conclusion is that—from the three analyzed solvation shells of CfAFP—the dynamical properties of solvation water of the ice-binding plane resemble the most the properties of solvation water of ice; note, however, that these properties still clearly differ from the dynamic properties of solvation water of ice.

  18. Animal ice-binding (antifreeze) proteins and glycolipids: an overview with emphasis on physiological function.

    PubMed

    Duman, John G

    2015-06-01

    Ice-binding proteins (IBPs) assist in subzero tolerance of multiple cold-tolerant organisms: animals, plants, fungi, bacteria etc. IBPs include: (1) antifreeze proteins (AFPs) with high thermal hysteresis antifreeze activity; (2) low thermal hysteresis IBPs; and (3) ice-nucleating proteins (INPs). Several structurally different IBPs have evolved, even within related taxa. Proteins that produce thermal hysteresis inhibit freezing by a non-colligative mechanism, whereby they adsorb onto ice crystals or ice-nucleating surfaces and prevent further growth. This lowers the so-called hysteretic freezing point below the normal equilibrium freezing/melting point, producing a difference between the two, termed thermal hysteresis. True AFPs with high thermal hysteresis are found in freeze-avoiding animals (those that must prevent freezing, as they die if frozen) especially marine fish, insects and other terrestrial arthropods where they function to prevent freezing at temperatures below those commonly experienced by the organism. Low thermal hysteresis IBPs are found in freeze-tolerant organisms (those able to survive extracellular freezing), and function to inhibit recrystallization - a potentially damaging process whereby larger ice crystals grow at the expense of smaller ones - and in some cases, prevent lethal propagation of extracellular ice into the cytoplasm. Ice-nucleator proteins inhibit supercooling and induce freezing in the extracellular fluid at high subzero temperatures in many freeze-tolerant species, thereby allowing them to control the location and temperature of ice nucleation, and the rate of ice growth. Numerous nuances to these functions have evolved. Antifreeze glycolipids with significant thermal hysteresis activity were recently identified in insects, frogs and plants.

  19. Significance of conservative asparagine residues in the thermal hysteresis activity of carrot antifreeze protein.

    PubMed Central

    Zhang, Dang-Quan; Liu, Bing; Feng, Dong-Ru; He, Yan-Ming; Wang, Shu-Qi; Wang, Hong-Bin; Wang, Jin-Fa

    2004-01-01

    The approximately 24-amino-acid leucine-rich tandem repeat motif (PXXXXXLXXLXXLXLSXNXLXGXI) of carrot antifreeze protein comprises most of the processed protein and should contribute at least partly to the ice-binding site. Structural predictions using publicly available online sources indicated that the theoretical three-dimensional model of this plant protein includes a 10-loop beta-helix containing the approximately 24-amino-acid tandem repeat. This theoretical model indicated that conservative asparagine residues create putative ice-binding sites with surface complementarity to the 1010 prism plane of ice. We used site-specific mutagenesis to test the importance of these residues, and observed a distinct loss of thermal hysteresis activity when conservative asparagines were replaced with valine or glutamine, whereas a large increase in thermal hysteresis was observed when phenylalanine or threonine residues were replaced with asparagine, putatively resulting in the formation of an ice-binding site. These results confirmed that the ice-binding site of carrot antifreeze protein consists of conservative asparagine residues in each beta-loop. We also found that its thermal hysteresis activity is directly correlated with the length of its asparagine-rich binding site, and hence with the size of its ice-binding face. PMID:14531728

  20. Activity of a Two-Domain Antifreeze Protein Is Not Dependent on Linker Sequence

    PubMed Central

    Holland, Nolan B.; Nishimiya, Yoshiyuki; Tsuda, Sakae; Sönnichsen, Frank D.

    2007-01-01

    The reported NMR structure of RD3, a naturally occurring two-domain antifreeze protein, suggests that the two nearly identical domains are oriented to allow simultaneous binding of their active regions to the ice surface. It is implied that the nine residues linking the two domains play a role in this alignment, but this has not been established. We have designed and expressed a modified form of RD3 that replaces the nine-residue linker with a generic sequence of one serine and eight glycine residues to test the importance of the linker amino acid sequence. The modified linker is shown to have significantly different characteristics compared to the original linker. Heteronuclear nuclear Overhauser effect experiments show that the new linker residues have more mobility than the linker residues in the native protein. Further, NMR data show that the folding of the C-terminal domain is somewhat perturbed by the altered linker. Finally, distributions of residual dipolar couplings indicate that the two domains tumble and move independently of each other. Nevertheless, the thermal hysteresis activity of the modified protein is indistinguishable from that of native RD3, proving that increased activity of the two-domain antifreeze protein is not dependent on structure of the linker. PMID:17056724

  1. Extraction, purification and identification of antifreeze proteins from cold acclimated malting barley (Hordeum vulgare L.).

    PubMed

    Ding, Xiangli; Zhang, Hui; Chen, Haiying; Wang, Li; Qian, Haifeng; Qi, Xiguang

    2015-05-15

    Antifreeze proteins from cold-acclimated malting barley were extracted by infiltration-centrifugation. The infiltration time was optimised, and its extraction effect was evaluated. The effect of cold acclimation on the accumulation of barley antifreeze proteins (BaAFPs) was assessed by comparing the thermal hysteresis activities (THA) of proteins extracted from both cold acclimated and non-cold acclimated barley grain. Ultra-filtration, ammonium precipitation and column chromatography were used successively to purify the BaAFPs, and MALDI-TOF-MS/MS was used for protein identification. The results showed that infiltration-centrifugation was more targeted than the traditional method, and 10h was the optimal infiltration time. THA was observed only after cold acclimation implied that AFPs only began to accumulate after cold acclimation. After purification, BaAFP-I was obtained at an electrophoresis level and its THA was 1.04°C (18.0 mg ml(-1)). The mass fingerprinting and sequencing results indicated the homology of the BaAFP-I to alpha-amylase inhibitor BDAI-1 (Hordeum vulgare).

  2. When are antifreeze proteins in solution essential for ice growth inhibition?

    PubMed

    Drori, Ran; Davies, Peter L; Braslavsky, Ido

    2015-06-02

    Antifreeze proteins (AFPs) are a widespread class of proteins that bind to ice and facilitate the survival of organisms under freezing conditions. AFPs have enormous potential in applications that require control over ice growth. However, the nature of the binding interaction between AFPs and ice remains the subject of debate. Using a microfluidics system developed in-house we previously showed that hyperactive AFP from the Tenebrio molitor beetle, TmAFP, remains bound to an ice crystal surface after exchanging the solution surrounding the ice crystal to an AFP-free solution. Furthermore, these surface-adsorbed TmAFP molecules sufficed to prevent ice growth. These experiments provided compelling evidence for the irreversible binding of hyperactive AFPs to ice. Here, we tested a moderately active type III AFP (AFPIII) from a fish in a similar microfluidics system. We found, in solution exchange experiments that the AFPIIIs were also irreversibly bound to the ice crystals. However, some crystals displayed "burst" growth during the solution exchange. AFPIII, like other moderately active fish AFPs, is unable to bind to the basal plane of an ice crystal. We showed that although moderate AFPs bound to ice irreversibly, moderate AFPs in solution were needed to inhibit ice growth from the bipyramidal crystal tips. Instead of binding to the basal plane, these AFPs minimized the basal face size by stabilizing other crystal planes that converge to form the crystal tips. Furthermore, when access of solution to the basal plane was physically blocked by the microfluidics device walls, we observed enhancement of the antifreeze activity. These findings provide direct evidence that the weak point of ice growth inhibition by fish AFPs is the basal plane, whereas insect AFPs, which can bind to the basal plane, are able to inhibit its growth and thereby increase antifreeze activity.

  3. Myeloid thrombomodulin lectin-like domain inhibits osteoclastogenesis and inflammatory bone loss.

    PubMed

    Cheng, Tsung-Lin; Lai, Chao-Han; Shieh, Shyh-Jou; Jou, Yin-Bo; Yeh, Jwu-Lai; Yang, Ai-Lun; Wang, Yan-Hsiung; Wang, Chau-Zen; Chen, Chung-Hwan; Shi, Guey-Yueh; Ho, Mei-Ling; Wu, Hua-Lin

    2016-06-17

    Osteoclastogenesis is an essential process during bone metabolism which can also be promoted by inflammatory signals. Thrombomodulin (TM), a transmembrane glycoprotein, exerts anti-inflammatory activities such as neutralization of proinflammatory high-mobility group box 1 (HMGB1) through TM lectin-like domain. This study aimed to identify the role of myeloid TM (i.e., endogenous TM expression on the myeloid lineage) in osteoclastogenesis and inflammatory bone loss. Using human peripheral blood mononuclear cells and mouse bone marrow-derived macrophages, we observed that the protein levels of TM were dramatically reduced as these cells differentiated into osteoclasts. In addition, osteoclastogenesis and extracellular HMGB1 accumulation were enhanced in primary cultured monocytes from myeloid-specific TM-deficient mice (LysMcre/TM(flox/flox)) and from TM lectin-like domain deleted mice (TM(LeD/LeD)) compared with their respective controls. Micro-computerized tomography scans showed that ovariectomy-induced bone loss was more pronounced in TM(LeD/LeD) mice compared with controls. Finally, the inhibiting effects of recombinant TM lectin-like domain (rTMD1) on bone resorption in vitro, and bone loss in both the ovariectomized model and collagen antibody-induced arthritis model has been detected. These findings suggested that the myeloid TM lectin-like domain may inhibit osteoclastogenesis by reducing HMGB1 signaling, and rTMD1 may hold therapeutic potential for inflammatory bone loss.

  4. Molecular cloning, sequence analysis and homology modeling of the first caudata amphibian antifreeze-like protein in axolotl (Ambystoma mexicanum).

    PubMed

    Zhang, Songyan; Gao, Jiuxiang; Lu, Yiling; Cai, Shasha; Qiao, Xue; Wang, Yipeng; Yu, Haining

    2013-08-01

    Antifreeze proteins (AFPs) refer to a class of polypeptides that are produced by certain vertebrates, plants, fungi, and bacteria and which permit their survival in subzero environments. In this study, we report the molecular cloning, sequence analysis and three-dimensional structure of the axolotl antifreeze-like protein (AFLP) by homology modeling of the first caudate amphibian AFLP. We constructed a full-length spleen cDNA library of axolotl (Ambystoma mexicanum). An EST having highest similarity (∼42%) with freeze-responsive liver protein Li16 from Rana sylvatica was identified, and the full-length cDNA was subsequently obtained by RACE-PCR. The axolotl antifreeze-like protein sequence represents an open reading frame for a putative signal peptide and the mature protein composed of 93 amino acids. The calculated molecular mass and the theoretical isoelectric point (pl) of this mature protein were 10128.6 Da and 8.97, respectively. The molecular characterization of this gene and its deduced protein were further performed by detailed bioinformatics analysis. The three-dimensional structure of current AFLP was predicted by homology modeling, and the conserved residues required for functionality were identified. The homology model constructed could be of use for effective drug design. This is the first report of an antifreeze-like protein identified from a caudate amphibian.

  5. Formation of ice-like water structure on the surface of an antifreeze protein.

    PubMed

    Smolin, Nikolai; Daggett, Valerie

    2008-05-15

    Antifreeze proteins (AFPs) are found in different species from polar, alpine, and subarctic regions where they serve to inhibit ice crystal growth by adsorption to ice surfaces. Computational methods have the power to investigate the antifreeze mechanism in atomic detail. Molecular dynamics simulations of water under different conditions have been carried out to test our water model for simulations of biological macromolecules in extreme conditions: very low temperatures (200 K) and at the ice/liquid water interface. We show that the flexible F3C water model reproduces properties of water in the solid phase (ice I(h)), the supercooled liquid phase, and at the ice/liquid water interface. Additionally, the hydration of the type III AFP from ocean pout was studied as a function of temperature. Hydration waters on the ice-binding surface of the AFP were less distorted and more tetrahedral than elsewhere on the surface. More ice-like hydrating water structures formed on the ice-binding surface of the protein such that it created an ice-like structure in water within its first hydration layer but not beyond, suggesting that this portion of the protein has high affinity for ice surfaces.

  6. Structure and interactions of fish type III antifreeze protein in solution.

    PubMed

    Salvay, Andrés G; Gabel, Frank; Pucci, Bernard; Santos, Javier; Howard, Eduardo I; Ebel, Christine

    2010-07-21

    It has been suggested that above a critical protein concentration, fish Type III antifreeze protein (AFP III) self-assembles to form micelle-like structures that may play a key role in antifreeze activity. To understand the complex activity of AFP III, a comprehensive description of its association state and structural organization in solution is necessary. We used analytical ultracentrifugation, analytical size-exclusion chromatography, and dynamic light scattering to characterize the interactions and homogeneity of AFP III in solution. Small-angle neutron scattering was used to determine the low-resolution structure in solution. Our results clearly show that at concentrations up to 20 mg mL(-1) and at temperatures of 20 degrees C, 6 degrees C, and 4 degrees C, AFP III is monomeric in solution and adopts a structure compatible with that determined by crystallography. Surface tension measurements show a propensity of AFP III to localize at the air/water interface, but this surface activity is not correlated with any aggregation in the bulk. These results support the hypothesis that each AFP III molecule acts independently of the others, and that specific intermolecular interactions between monomers are not required for binding to ice. The lack of attractive interactions between monomers may be functionally important, allowing for more efficient binding and covering of the ice surface.

  7. Ice-surface adsorption enhanced colligative effect of antifreeze proteins in ice growth inhibition

    NASA Astrophysics Data System (ADS)

    Mao, Yougang; Ba, Yong

    2006-09-01

    This Communication describes a mechanism to explain antifreeze protein's function to inhibit the growth of ice crystals. We propose that the adsorption of antifreeze protein (AFP) molecules on an ice surface induces a dense AFP-water layer, which can significantly decrease the mole fraction of the interfacial water and, thus, lower the temperature for a seed ice crystal to grow in a super-cooled AFP solution. This mechanism can also explain the nearly unchanged melting point for the ice crystal due to the AFP's ice-surface adsorption. A mathematical model combining the Langmuir theory of adsorption and the colligative effect of thermodynamics has been proposed to find the equilibrium constants of the ice-surface adsorptions, and the interfacial concentrations of AFPs through fitting the theoretical curves to the experimental thermal hysteresis data. This model has been demonstrated by using the experimental data of serial size-mutated beetle Tenebrio molitor (Tm) AFPs. It was found that the AFP's ice-surface adsorptions could increase the interfacial AFP's concentrations by 3 to 4 orders compared with those in the bulk AFP solutions.

  8. Ice-surface adsorption enhanced colligative effect of antifreeze proteins in ice growth inhibition.

    PubMed

    Mao, Yougang; Ba, Yong

    2006-09-07

    This Communication describes a mechanism to explain antifreeze protein's function to inhibit the growth of ice crystals. We propose that the adsorption of antifreeze protein (AFP) molecules on an ice surface induces a dense AFP-water layer, which can significantly decrease the mole fraction of the interfacial water and, thus, lower the temperature for a seed ice crystal to grow in a super-cooled AFP solution. This mechanism can also explain the nearly unchanged melting point for the ice crystal due to the AFP's ice-surface adsorption. A mathematical model combining the Langmuir theory of adsorption and the colligative effect of thermodynamics has been proposed to find the equilibrium constants of the ice-surface adsorptions, and the interfacial concentrations of AFPs through fitting the theoretical curves to the experimental thermal hysteresis data. This model has been demonstrated by using the experimental data of serial size-mutated beetle Tenebrio molitor (Tm) AFPs. It was found that the AFP's ice-surface adsorptions could increase the interfacial AFP's concentrations by 3 to 4 orders compared with those in the bulk AFP solutions.

  9. Structure and Interactions of Fish Type III Antifreeze Protein in Solution

    PubMed Central

    Salvay, Andrés G.; Gabel, Frank; Pucci, Bernard; Santos, Javier; Howard, Eduardo I.; Ebel, Christine

    2010-01-01

    Abstract It has been suggested that above a critical protein concentration, fish Type III antifreeze protein (AFP III) self-assembles to form micelle-like structures that may play a key role in antifreeze activity. To understand the complex activity of AFP III, a comprehensive description of its association state and structural organization in solution is necessary. We used analytical ultracentrifugation, analytical size-exclusion chromatography, and dynamic light scattering to characterize the interactions and homogeneity of AFP III in solution. Small-angle neutron scattering was used to determine the low-resolution structure in solution. Our results clearly show that at concentrations up to 20 mg mL−1 and at temperatures of 20°C, 6°C, and 4°C, AFP III is monomeric in solution and adopts a structure compatible with that determined by crystallography. Surface tension measurements show a propensity of AFP III to localize at the air/water interface, but this surface activity is not correlated with any aggregation in the bulk. These results support the hypothesis that each AFP III molecule acts independently of the others, and that specific intermolecular interactions between monomers are not required for binding to ice. The lack of attractive interactions between monomers may be functionally important, allowing for more efficient binding and covering of the ice surface. PMID:20643081

  10. Systematic size study of an insect antifreeze protein and its interaction with ice.

    PubMed

    Liu, Kai; Jia, Zongchao; Chen, Guangju; Tung, Chenho; Liu, Ruozhuang

    2005-02-01

    Because of their remarkable ability to depress the freezing point of aqueous solutions, antifreeze proteins (AFPs) play a critical role in helping many organisms survive subzero temperatures. The beta-helical insect AFP structures solved to date, consisting of multiple repeating circular loops or coils, are perhaps the most regular protein structures discovered thus far. Taking an exceptional advantage of the unusually high structural regularity of insect AFPs, we have employed both semiempirical and quantum mechanics computational approaches to systematically investigate the relationship between the number of AFP coils and the AFP-ice interaction energy, an indicator of antifreeze activity. We generated a series of AFP models with varying numbers of 12-residue coils (sequence TCTxSxxCxxAx) and calculated their interaction energies with ice. Using several independent computational methods, we found that the AFP-ice interaction energy increased as the number of coils increased, until an upper bound was reached. The increase of interaction energy was significant for each of the first five coils, and there was a clear synergism that gradually diminished and even decreased with further increase of the number of coils. Our results are in excellent agreement with the recently reported experimental observations.

  11. The inhibition of tetrahydrofuran clathrate-hydrate formation with antifreeze protein

    NASA Astrophysics Data System (ADS)

    Zeng, H.; Wilson, L. D.; Walker, V. K.; Ripmeester, J. A.

    2003-01-01

    The effect of Type I fish antifreeze protein (AFP) from the winter flounder, Pleuronectes americanus (Walbaum), (WfAFP) on the formation of tetrahydrofuran (THF) clathrate hydrate was studied by observing changes in THF crystal morphology and determining the induction time for nucleation. AFP retarded THF clathrate-hydrate growth at the tested temperatures and modified the THF clathrate-hydrate crystal morphology from octahedral to plate-like. AFP appears to be even more effective than the kinetic inhibitor, polyvinylpyrrolidone (PVP). Recombinant AFP from an insect, a spruce budworm, Choristoneura fumiferana (Clem.), moth, (Cf) was also tested for inhibition activity by observation of the THF-hydrate-crystal-growth habit. Like WfAFP, CfAFP appeared to show adsorption on multiple THF-hydrate-crystal faces. A protein with no antifreeze activity, cytochrome C, was used as a control and it neither changed the morphology of the THF clathrate-hydrate crystals, nor retarded the formation of the hydrate. Preliminary experiments on the inhibition activity of WfAFP on a natural gas hydrate assessed induction time and the amount of propane gas consumed. Similar to the observations for THF, the data indicated that WfAFP inhibited propane-hydrate growth. Taken together, these results support our hypothesis that AFPs can inhibit clathrate-hydrate growth and as well, offer promise for the understanding of the inhibition mechanism.

  12. Induced ice melting by the snow flea antifreeze protein from molecular dynamics simulations.

    PubMed

    Todde, Guido; Whitman, Christopher; Hovmöller, Sven; Laaksonen, Aatto

    2014-11-26

    Antifreeze proteins (AFP) allow different life forms, insects as well as fish and plants, to survive in subzero environments. AFPs prevent freezing of the physiological fluids. We have studied, through molecular dynamics simulations, the behavior of the small isoform of the AFP found in the snow flea (sfAFP), both in water and at the ice/water interface, of four different ice planes. In water at room temperature, the structure of the sfAFP is found to be slightly unstable. The loop between two polyproline II helices has large fluctuations as well as the C-terminus. Torsional angle analyses show a decrease of the polyproline II helix area in the Ramachandran plots. The protein structure instability, in any case, should not affect its antifreeze activity. At the ice/water interface the sfAFP triggers local melting of the ice surface. Bipyramidal, secondary prism, and prism ice planes melt in the presence of AFP at temperatures below the melting point of ice. Only the basal plane is found to be stable at the same temperatures, indicating an adsorption of the sfAFP on this ice plane as confirmed by experimental evidence.

  13. Hybridization assay of insect antifreezing protein gene by novel multilayered porous silicon nucleic acid biosensor.

    PubMed

    Lv, Xiaoyi; Chen, Liangliang; Zhang, Hongyan; Mo, Jiaqing; Zhong, Furu; Lv, Changwu; Ma, Ji; Jia, Zhenhong

    2013-01-15

    A fabrication of a novel simple porous silicon polybasic photonic crystal with symmetrical structure has been reported as a nucleic acid biosensor for detecting antifreeze protein gene in insects (Microdera puntipennis dzhungarica), which would be helpful in the development of some new transgenic plants with tolerance of freezing stress. Compared to various porous silicon-based photonic configurations, porous silicon polytype layered structure is quite easy to prepare and shows more stability; moreover, polybasic photonic crystals with symmetrical structure exhibit interesting optical properties with a sharp resonance in the reflectance spectrum, giving a higher Q factor which causes higher sensitivity for sensing performance. In this experiment, DNA oligonucleotides were immobilized into the porous silicon pores using a standard crosslink chemistry method. The porous silicon polybasic symmetrical structure sensor possesses high specificity in performing controlled experiments with non-complementary DNA. The detection limit was found to be 21.3nM for DNA oligonucleotides. The fabricated multilayered porous silicon-based DNA biosensor has potential commercial applications in clinical chemistry for determination of an antifreeze protein gene or other genes.

  14. The biological function of an insect antifreeze protein simulated by molecular dynamics

    PubMed Central

    Kuiper, Michael J; Morton, Craig J; Abraham, Sneha E; Gray-Weale, Angus

    2015-01-01

    Antifreeze proteins (AFPs) protect certain cold-adapted organisms from freezing to death by selectively adsorbing to internal ice crystals and inhibiting ice propagation. The molecular details of AFP adsorption-inhibition is uncertain but is proposed to involve the Gibbs–Thomson effect. Here we show by using unbiased molecular dynamics simulations a protein structure-function mechanism for the spruce budworm Choristoneura fumiferana AFP, including stereo-specific binding and consequential melting and freezing inhibition. The protein binds indirectly to the prism ice face through a linear array of ordered water molecules that are structurally distinct from the ice. Mutation of the ice binding surface disrupts water-ordering and abolishes activity. The adsorption is virtually irreversible, and we confirm the ice growth inhibition is consistent with the Gibbs–Thomson law. DOI: http://dx.doi.org/10.7554/eLife.05142.001 PMID:25951514

  15. Creating Anti-icing Surfaces via the Direct Immobilization of Antifreeze Proteins on Aluminum

    PubMed Central

    Gwak, Yunho; Park, Ji-in; Kim, Minjae; Kim, Hong Suk; Kwon, Myong Jong; Oh, Seung Jin; Kim, Young-Pil; Jin, EonSeon

    2015-01-01

    Cryoprotectants such as antifreeze proteins (AFPs) and sugar molecules may provide a solution for icing problems. These anti-icing substances protect cells and tissues from freezing by inhibiting ice formation. In this study, we developed a method for coating an industrial metal material (aluminum, Al) with AFP from the Antarctic marine diatom, Chaetoceros neogracile (Cn-AFP), to prevent or delay ice formation. To coat Al with Cn-AFP, we used an Al-binding peptide (ABP) as a conjugator and fused it with Cn-AFP. The ABP bound well to the Al and did not considerably change the functional properties of AFP. Cn-AFP-coated Al (Cn-AFP-Al) showed a sufficiently low supercooling point. Additional trehalose coating of Cn-AFP-Al considerably delayed AFP denaturation on the Al without affecting its antifreeze activity. This metal surface–coating method using trehalose-fortified AFP can be applied to other metals important in the aircraft and cold storage fields where anti-icing materials are critical. PMID:26153855

  16. Creating Anti-icing Surfaces via the Direct Immobilization of Antifreeze Proteins on Aluminum.

    PubMed

    Gwak, Yunho; Park, Ji-in; Kim, Minjae; Kim, Hong Suk; Kwon, Myong Jong; Oh, Seung Jin; Kim, Young-Pil; Jin, EonSeon

    2015-07-08

    Cryoprotectants such as antifreeze proteins (AFPs) and sugar molecules may provide a solution for icing problems. These anti-icing substances protect cells and tissues from freezing by inhibiting ice formation. In this study, we developed a method for coating an industrial metal material (aluminum, Al) with AFP from the Antarctic marine diatom, Chaetoceros neogracile (Cn-AFP), to prevent or delay ice formation. To coat Al with Cn-AFP, we used an Al-binding peptide (ABP) as a conjugator and fused it with Cn-AFP. The ABP bound well to the Al and did not considerably change the functional properties of AFP. Cn-AFP-coated Al (Cn-AFP-Al) showed a sufficiently low supercooling point. Additional trehalose coating of Cn-AFP-Al considerably delayed AFP denaturation on the Al without affecting its antifreeze activity. This metal surface-coating method using trehalose-fortified AFP can be applied to other metals important in the aircraft and cold storage fields where anti-icing materials are critical.

  17. An Effective Antifreeze Protein Predictor with Ensemble Classifiers and Comprehensive Sequence Descriptors

    PubMed Central

    Yang, Runtao; Zhang, Chengjin; Gao, Rui; Zhang, Lina

    2015-01-01

    Antifreeze proteins (AFPs) play a pivotal role in the antifreeze effect of overwintering organisms. They have a wide range of applications in numerous fields, such as improving the production of crops and the quality of frozen foods. Accurate identification of AFPs may provide important clues to decipher the underlying mechanisms of AFPs in ice-binding and to facilitate the selection of the most appropriate AFPs for several applications. Based on an ensemble learning technique, this study proposes an AFP identification system called AFP-Ensemble. In this system, random forest classifiers are trained by different training subsets and then aggregated into a consensus classifier by majority voting. The resulting predictor yields a sensitivity of 0.892, a specificity of 0.940, an accuracy of 0.938 and a balanced accuracy of 0.916 on an independent dataset, which are far better than the results obtained by previous methods. These results reveal that AFP-Ensemble is an effective and promising predictor for large-scale determination of AFPs. The detailed feature analysis in this study may give useful insights into the molecular mechanisms of AFP-ice interactions and provide guidance for the related experimental validation. A web server has been designed to implement the proposed method. PMID:26370959

  18. Structural basis for antifreeze activity of ice-binding protein from arctic yeast.

    PubMed

    Lee, Jun Hyuck; Park, Ae Kyung; Do, Hackwon; Park, Kyoung Sun; Moh, Sang Hyun; Chi, Young Min; Kim, Hak Jun

    2012-03-30

    Arctic yeast Leucosporidium sp. produces a glycosylated ice-binding protein (LeIBP) with a molecular mass of ∼25 kDa, which can lower the freezing point below the melting point once it binds to ice. LeIBP is a member of a large class of ice-binding proteins, the structures of which are unknown. Here, we report the crystal structures of non-glycosylated LeIBP and glycosylated LeIBP at 1.57- and 2.43-Å resolution, respectively. Structural analysis of the LeIBPs revealed a dimeric right-handed β-helix fold, which is composed of three parts: a large coiled structural domain, a long helix region (residues 96-115 form a long α-helix that packs along one face of the β-helix), and a C-terminal hydrophobic loop region ((243)PFVPAPEVV(251)). Unexpectedly, the C-terminal hydrophobic loop region has an extended conformation pointing away from the body of the coiled structural domain and forms intertwined dimer interactions. In addition, structural analysis of glycosylated LeIBP with sugar moieties attached to Asn(185) provides a basis for interpreting previous biochemical analyses as well as the increased stability and secretion of glycosylated LeIBP. We also determined that the aligned Thr/Ser/Ala residues are critical for ice binding within the B face of LeIBP using site-directed mutagenesis. Although LeIBP has a common β-helical fold similar to that of canonical hyperactive antifreeze proteins, the ice-binding site is more complex and does not have a simple ice-binding motif. In conclusion, we could identify the ice-binding site of LeIBP and discuss differences in the ice-binding modes compared with other known antifreeze proteins and ice-binding proteins.

  19. Why ice-binding type I antifreeze protein acts as a gas hydrate crystal inhibitor.

    PubMed

    Bagherzadeh, S Alireza; Alavi, Saman; Ripmeester, John A; Englezos, Peter

    2015-04-21

    Antifreeze proteins (AFPs) prevent ice growth by binding to a specific ice plane. Some AFPs have been found to inhibit the formation of gas hydrates which are a serious safety and operational challenge for the oil and gas industry. Molecular dynamics simulations are used to determine the mechanism of action of the winter flounder AFP (wf-AFP) in inhibiting methane hydrate growth. The wf-AFP adsorbs onto the methane hydrate surface via cooperative binding of a set of hydrophobic methyl pendant groups to the empty half-cages at the hydrate/water interface. Each binding set is composed of the methyl side chain of threonine and two alanine residues, four and seven places further down in the sequence of the protein. Understanding the principle of action of AFPs can lead to the rational design of green hydrate inhibitor molecules with potential superior performance.

  20. Solution structure of an antifreeze protein CfAFP-501 from Choristoneura fumiferana.

    PubMed

    Li, Congmin; Guo, Xianrong; Jia, Zongchao; Xia, Bin; Jin, Changwen

    2005-07-01

    Antifreeze proteins (AFPs) are widely employed by various organisms as part of their overwintering survival strategy. AFPs have the unique ability to suppress the freezing point of aqueous solution and inhibit ice recrystallization through binding to the ice seed crystals and restricting their growth. The solution structure of CfAFP-501 from spruce budworm has been determined by NMR spectroscopy. Our result demonstrates that CfAFP-501 retains its rigid and highly regular structure in solution. Overall, the solution structure is similar to the crystal structure except the N- and C-terminal regions. NMR spin-relaxation experiments further indicate the overall rigidity of the protein and identify a collection of residues with greater flexibilities. Furthermore, Pro91 shows a cis conformation in solution instead of the trans conformation determined in the crystal structure.

  1. High water mobility on the ice-binding surface of a hyperactive antifreeze protein.

    PubMed

    Modig, Kristofer; Qvist, Johan; Marshall, Christopher B; Davies, Peter L; Halle, Bertil

    2010-09-21

    Antifreeze proteins (AFPs) prevent uncontrolled ice formation in organisms exposed to subzero temperatures by binding irreversibly to specific planes of nascent ice crystals. To understand the thermodynamic driving forces and kinetic mechanism of AFP activity, it is necessary to characterize the hydration behavior of these proteins in solution. With this aim, we have studied the hyperactive insect AFP from Tenebrio molitor (TmAFP) with the (17)O magnetic relaxation dispersion (MRD) method, which selectively monitors the rotational motion and exchange kinetics of water molecules on picosecond-microsecond time scales. The global hydration behavior of TmAFP is found to be similar to non-antifreeze proteins, with no evidence of ice-like or long-ranged modifications of the solvent. However, two sets of structural water molecules, located within the core and on the ice-binding face in the crystal structure of TmAFP, may have functional significance. We find that 2 of the 5 internal water molecules exchange with a residence time of 8 +/- 1 micros at 300 K and a large activation energy of approximately 50 kJ mol(-1), reflecting intermittent large-scale conformational fluctuations in this exceptionally dense and rigid protein. Six water molecules arrayed with ice-like spacing in the central trough on the ice-binding face exchange with bulk water on a sub-nanosecond time scale. The combination of high order and fast exchange may allow these water molecules to contribute entropically to the ice-binding affinity without limiting the absorption rate.

  2. Crystal structure of an insect antifreeze protein and its implications for ice binding.

    PubMed

    Hakim, Aaron; Nguyen, Jennifer B; Basu, Koli; Zhu, Darren F; Thakral, Durga; Davies, Peter L; Isaacs, Farren J; Modis, Yorgo; Meng, Wuyi

    2013-04-26

    Antifreeze proteins (AFPs) help some organisms resist freezing by binding to ice crystals and inhibiting their growth. The molecular basis for how these proteins recognize and bind ice is not well understood. The longhorn beetle Rhagium inquisitor can supercool to below -25 °C, in part by synthesizing the most potent antifreeze protein studied thus far (RiAFP). We report the crystal structure of the 13-kDa RiAFP, determined at 1.21 Å resolution using direct methods. The structure, which contains 1,914 nonhydrogen protein atoms in the asymmetric unit, is the largest determined ab initio without heavy atoms. It reveals a compressed β-solenoid fold in which the top and bottom sheets are held together by a silk-like interdigitation of short side chains. RiAFP is perhaps the most regular structure yet observed. It is a second independently evolved AFP type in beetles. The two beetle AFPs have in common an extremely flat ice-binding surface comprising regular outward-projecting parallel arrays of threonine residues. The more active, wider RiAFP has four (rather than two) of these arrays between which the crystal structure shows the presence of ice-like waters. Molecular dynamics simulations independently reproduce the locations of these ordered crystallographic waters and predict additional waters that together provide an extensive view of the AFP interaction with ice. By matching several planes of hexagonal ice, these waters may help freeze the AFP to the ice surface, thus providing the molecular basis of ice binding.

  3. Antifreeze protein from shorthorn sculpin: identification of the ice-binding surface.

    PubMed

    Baardsnes, J; Jelokhani-Niaraki, M; Kondejewski, L H; Kuiper, M J; Kay, C M; Hodges, R S; Davies, P L

    2001-12-01

    Shorthorn sculpins, Myoxocephalus scorpius, are protected from freezing in icy seawater by alanine-rich, alpha-helical antifreeze proteins (AFPs). The major serum isoform (SS-8) has been reisolated and analyzed to establish its correct sequence. Over most of its length, this 42 amino acid protein is predicted to be an amphipathic alpha-helix with one face entirely composed of Ala residues. The other side of the helix, which is more heterogeneous and hydrophilic, contains several Lys. Computer simulations had suggested previously that these Lys residues were involved in binding of the peptide to the [11-20] plane of ice in the <-1102> direction. To test this hypothesis, a series of SS-8 variants were generated with single Ala to Lys substitutions at various points around the helix. All of the peptides retained significant alpha-helicity and remained as monomers in solution. Substitutions on the hydrophilic helix face at position 16, 19, or 22 had no obvious effect, but those on the adjacent Ala-rich surface at positions 17, 21, and 25 abolished antifreeze activity. These results, with support from our own modeling and docking studies, show that the helix interacts with the ice surface via the conserved alanine face, and lend support to the emerging idea that the interaction of fish AFPs with ice involves appreciable hydrophobic interactions. Furthermore, our modeling suggests a new N terminus cap structure, which helps to stabilize the helix, whereas the role of the lysines on the hydrophilic face may be to enhance solubility of the protein.

  4. Re-Evaluation of a Bacterial Antifreeze Protein as an Adhesin with Ice-Binding Activity

    PubMed Central

    Guo, Shuaiqi; Garnham, Christopher P.; Whitney, John C.; Graham, Laurie A.; Davies, Peter L.

    2012-01-01

    A novel role for antifreeze proteins (AFPs) may reside in an exceptionally large 1.5-MDa adhesin isolated from an Antarctic Gram-negative bacterium, Marinomonas primoryensis. MpAFP was purified from bacterial lysates by ice adsorption and gel electrophoresis. We have previously reported that two highly repetitive sequences, region II (RII) and region IV (RIV), divide MpAFP into five distinct regions, all of which require mM Ca2+ levels for correct folding. Also, the antifreeze activity is confined to the 322-residue RIV, which forms a Ca2+-bound beta-helix containing thirteen Repeats-In-Toxin (RTX)-like repeats. RII accounts for approximately 90% of the mass of MpAFP and is made up of ∼120 tandem 104-residue repeats. Because these repeats are identical in DNA sequence, their number was estimated here by pulsed-field gel electrophoresis. Structural homology analysis by the Protein Homology/analogY Recognition Engine (Phyre2) server indicates that the 104-residue RII repeat adopts an immunoglobulin beta-sandwich fold that is typical of many secreted adhesion proteins. Additional RTX-like repeats in RV may serve as a non-cleavable signal sequence for the type I secretion pathway. Immunodetection shows both repeated regions are uniformly distributed over the cell surface. We suggest that the development of an AFP-like domain within this adhesin attached to the bacterial outer surface serves to transiently bind the host bacteria to ice. This association would keep the bacteria within the upper reaches of the water column where oxygen and nutrients are potentially more abundant. This novel envirotactic role would give AFPs a third function, after freeze avoidance and freeze tolerance: that of transiently binding an organism to ice. PMID:23144980

  5. Molecular Recognition of Methyl α-d-Mannopyranoside by Antifreeze (Glyco)Proteins

    PubMed Central

    2015-01-01

    Antifreeze proteins and glycoproteins [AF(G)Ps] have been well-known for more than three decades for their ability to inhibit the growth and recrystallization of ice through binding to specific ice crystal faces, and they show remarkable structural compatibility with specific ice crystal faces. Here, we show that the crystal growth faces of methyl α-d-mannopyranoside (MDM), a representative pyranose sugar, also show noteworthy structural compatibility with the known periodicities of AF(G)Ps. We selected fish AFGPs (AFGP8, AFGP1–5), and a beetle AFP (DAFP1) with increasing antifreeze activity as potential additives for controlling MDM crystal growth. Similar to their effects on ice growth, the AF(G)Ps can inhibit MDM crystal growth and recrystallization, and more significantly, the effectiveness for the AF(G)Ps are well correlated with their antifreeze activity. MDM crystals grown in the presence of AF(G)Ps are smaller and have better defined shapes and are of higher quality as indicated by single crystal X-ray diffraction and polarized microscopy than control crystals, but no new polymorphs of MDM were identified by single crystal X-ray diffraction, solid-state NMR, and attenuated total reflectance infrared spectroscopy. The observed changes in the average sizes of the MDM crystals can be related to the changes in the number of the MDM nuclei in the presence of the AF(G)Ps. The critical free energy change differences of the MDM nucleation in the absence and presence of the additives were calculated. These values are close to those of the ice nucleation in the presence of AF(G)Ps suggesting similar interactions are involved in the molecular recognition of MDM by the AF(G)Ps. To our knowledge this is the first report where AF(G)Ps have been used to control crystal growth of carbohydrates and on AFGPs controlling non-ice-like crystals. Our finding suggests MDM might be a possible alternative to ice for studying the detailed mechanism of AF

  6. The C-type lectin-like receptors of Dectin-1 cluster in natural killer gene complex.

    PubMed

    Xie, Jianhui

    2012-08-01

    Natural killer gene complex (NKC) encodes a group of proteins with a single C-type lectin-like domain, (CTLD) which can be subdivided several subfamilies according to their structures and expression patterns. The receptors containing the conserved calcium binding sites in the CTLD fold belong to group II of C-type lectin superfamily and are expressed on myeloid cells and non- myeloid cells. The receptors lacking conserved calcium binding sites in the CTLD fold have evolved to bind ligands other than carbohydrates independently on calcium and thereby are named as C-type lectin-like receptors. The C-type lectin-like receptors are previously thought to be exclusively expressed on natural killer (NK) cells and enable NK cells to discriminate self, missing self or altered self. However, some C-type lectin-like receptors are identified in myeloid cells and are intensely investigated, recently. These myeloid C-type lectin-like receptors, especially Dectin-1 cluster, have a wide variety of ligands, including those of exogenous origin, and play important roles in the physiological functions and pathological processes including immune homeostasis, immune defenses, and immune surveillance. In this review, we summarize each member of the Dectin-1 cluster, including their structural profiles, expression patterns, signaling properties as well as known physiological functions.

  7. Detecting seasonal variation of antifreeze protein distribution in Rhagium mordax using immunofluorescence and high resolution microscopy.

    PubMed

    Buch, J L; Ramløv, H

    2017-02-01

    Larvae of the blackspotted pliers support beetle, Rhagium mordax, were collected monthly, for the duration of 2012 and fixed. The larvae were embedded in paraffin wax and sectioned. Using fluorophore-coupled antibodies specific to the R. mordax antifreeze protein, RmAFP1, sections were visualised with UV reflected light microscopy. An automated software analysis method was developed in order to discard autofluorescence, and quantify fluorescence from bound antibodies. The results show that R. mordax cuticle and gut exhibit a higher degree of fluorophore-bound fluorescence during summer, than in the cold months. It is hypothesised that R. mordax stores RmAFP1 in, or near, the fat body during times when freeze avoidance is not needed.

  8. A hyperactive, Ca2+-dependent antifreeze protein in an Antarctic bacterium.

    PubMed

    Gilbert, Jack A; Davies, Peter L; Laybourn-Parry, Johanna

    2005-04-01

    In cold climates, some plants and bacteria that cannot avoid freezing use antifreeze proteins (AFPs) to lessen the destructive effects of ice recrystallization. These AFPs have weak freezing point depression activity, perhaps to avoid sudden, uncontrolled growth of ice. Here, we report on an uncharacteristically powerful bacterial AFP found in an Antarctic strain of the bacterium, Marinomonas primoryensis. It is Ca(2+)-dependent, shows evidence of cooperativity, and can produce over 2 degrees C of freezing point depression. Unlike most AFPs, it does not produce obvious crystal faceting during thermal hysteresis. This AFP might be capable of imparting freezing avoidance to M. primoryensis in ice-covered Antarctic lakes. A hyperactive bacterial AFP has not previously been reported.

  9. Fluorescence microscopy studies of the hyperactive antifreeze protein from an insect

    NASA Astrophysics Data System (ADS)

    Pertaya, N.; di Prinzio, C. L.; Wilen, L.; Thomson, E.; Wettlaufer, J. S.; Marshall, C. B.; Davies, P. L.; Braslavsky, I.

    2006-03-01

    Antifreeze proteins (AFPs) protect animals from freezing by binding to extracellular ice and inhibiting its growth. Since the initial discovery of AFPs in fish, non-homologous types have been found in insects, plants, bacteria, fungi, and vertebrates. Different AFP types have diverse structures and varied activities. For example, AFPs produced by insects are much more active in inhibiting ice crystal growth compared to most AFPs found in fish or plants. By putting a fluorescent tag on an insect AFP we were able to visualize AFP binding to ice, to determine the ice crystal surfaces to which the AFP adheres, and to follow the kinetics of AFP binding to ice. We expect this approach will contribute to a better understanding of the mechanism of AFP activity and in particular the hyperactivity of insect AFPs.

  10. Antifreeze proteins govern the precipitation of trehalose in a freezing-avoiding insect at low temperature.

    PubMed

    Wen, Xin; Wang, Sen; Duman, John G; Arifin, Josh Fnu; Juwita, Vonny; Goddard, William A; Rios, Alejandra; Liu, Fan; Kim, Soo-Kyung; Abrol, Ravinder; DeVries, Arthur L; Henling, Lawrence M

    2016-06-14

    The remarkable adaptive strategies of insects to extreme environments are linked to the biochemical compounds in their body fluids. Trehalose, a versatile sugar molecule, can accumulate to high levels in freeze-tolerant and freeze-avoiding insects, functioning as a cryoprotectant and a supercooling agent. Antifreeze proteins (AFPs), known to protect organisms from freezing by lowering the freezing temperature and deferring the growth of ice, are present at high levels in some freeze-avoiding insects in winter, and yet, paradoxically are found in some freeze-tolerant insects. Here, we report a previously unidentified role for AFPs in effectively inhibiting trehalose precipitation in the hemolymph (or blood) of overwintering beetle larvae. We determine the trehalose level (29.6 ± 0.6 mg/mL) in the larval hemolymph of a beetle, Dendroides canadensis, and demonstrate that the hemolymph AFPs are crucial for inhibiting trehalose crystallization, whereas the presence of trehalose also enhances the antifreeze activity of AFPs. To dissect the molecular mechanism, we examine the molecular recognition between AFP and trehalose crystal interfaces using molecular dynamics simulations. The theory corroborates the experiments and shows preferential strong binding of the AFP to the fast growing surfaces of the sugar crystal. This newly uncovered role for AFPs may help explain the long-speculated role of AFPs in freeze-tolerant species. We propose that the presence of high levels of molecules important for survival but prone to precipitation in poikilotherms (their body temperature can vary considerably) needs a companion mechanism to prevent the precipitation and here present, to our knowledge, the first example. Such a combination of trehalose and AFPs also provides a novel approach for cold protection and for trehalose crystallization inhibition in industrial applications.

  11. Antifreeze proteins govern the precipitation of trehalose in a freezing-avoiding insect at low temperature

    PubMed Central

    Wen, Xin; Wang, Sen; Duman, John G.; Arifin, Josh Fnu; Juwita, Vonny; Goddard, William A.; Rios, Alejandra; Liu, Fan; Kim, Soo-Kyung; Abrol, Ravinder; DeVries, Arthur L.; Henling, Lawrence M.

    2016-01-01

    The remarkable adaptive strategies of insects to extreme environments are linked to the biochemical compounds in their body fluids. Trehalose, a versatile sugar molecule, can accumulate to high levels in freeze-tolerant and freeze-avoiding insects, functioning as a cryoprotectant and a supercooling agent. Antifreeze proteins (AFPs), known to protect organisms from freezing by lowering the freezing temperature and deferring the growth of ice, are present at high levels in some freeze-avoiding insects in winter, and yet, paradoxically are found in some freeze-tolerant insects. Here, we report a previously unidentified role for AFPs in effectively inhibiting trehalose precipitation in the hemolymph (or blood) of overwintering beetle larvae. We determine the trehalose level (29.6 ± 0.6 mg/mL) in the larval hemolymph of a beetle, Dendroides canadensis, and demonstrate that the hemolymph AFPs are crucial for inhibiting trehalose crystallization, whereas the presence of trehalose also enhances the antifreeze activity of AFPs. To dissect the molecular mechanism, we examine the molecular recognition between AFP and trehalose crystal interfaces using molecular dynamics simulations. The theory corroborates the experiments and shows preferential strong binding of the AFP to the fast growing surfaces of the sugar crystal. This newly uncovered role for AFPs may help explain the long-speculated role of AFPs in freeze-tolerant species. We propose that the presence of high levels of molecules important for survival but prone to precipitation in poikilotherms (their body temperature can vary considerably) needs a companion mechanism to prevent the precipitation and here present, to our knowledge, the first example. Such a combination of trehalose and AFPs also provides a novel approach for cold protection and for trehalose crystallization inhibition in industrial applications. PMID:27226297

  12. Antifreeze poisoning

    MedlinePlus

    The poisonous ingredients in antifreeze are: Ethylene glycol Methanol Propylene glycol ... For ethylene glycol: Death may occur within the first 24 hours. If the patient survives, there may be little ...

  13. Fluorescence Microscopy Evidence for Quasi-Permanent Attachment of Antifreeze Proteins to Ice Surfaces

    PubMed Central

    Pertaya, Natalya; Marshall, Christopher B.; DiPrinzio, Carlos L.; Wilen, Larry; Thomson, Erik S.; Wettlaufer, J. S.; Davies, Peter L.; Braslavsky, Ido

    2007-01-01

    Many organisms are protected from freezing by the presence of extracellular antifreeze proteins (AFPs), which bind to ice, modify its morphology, and prevent its further growth. These proteins have a wide range of applications including cryopreservation, frost protection, and as models in biomineralization research. However, understanding their mechanism of action remains an outstanding challenge. While the prevailing adsorption-inhibition hypothesis argues that AFPs must bind irreversibly to ice to arrest its growth, other theories suggest that there is exchange between the bound surface proteins and the free proteins in solution. By conjugating green fluorescence protein (GFP) to a fish AFP (Type III), we observed the binding of the AFP to ice. This was accomplished by monitoring the presence of GFP-AFP on the surface of ice crystals several microns in diameter using fluorescence microscopy. The lack of recovery of fluorescence after photobleaching of the GFP component of the surface-bound GFP-AFP shows that there is no equilibrium surface-solution exchange of GFP-AFP and thus supports the adsorption-inhibition mechanism for this type of AFP. Moreover, our study establishes the utility of fluorescently labeled AFPs as a research tool for investigating the mechanisms underlying the activity of this diverse group of proteins. PMID:17325008

  14. Thrombomodulin-mediated cell adhesion: involvement of its lectin-like domain.

    PubMed

    Huang, Huey-Chun; Shi, Guey-Yueh; Jiang, Shinn-Jong; Shi, Chung-Sheng; Wu, Chun-Mei; Yang, Hsi-Yuan; Wu, Hua-Lin

    2003-11-21

    Thrombomodulin (TM) is an integral membrane glycoprotein that is a potent anticoagulant factor. TM may also possess functions distinct from its anticoagulant activity. Here the influence of TM on cell adhesion was studied in TM-negative melanoma A2058 cells transfected with green fluorescent protein-tagged TM (TMG) or lectin domain-deleted TM (TMG(DeltaL)). Confocal microscopy demonstrated that both TMG and TMG(DeltaL) were distributed in the plasma membrane. TMG-expressed cells grew as closely clustered colonies, with TM localized prominently in the intercellular boundaries. TMG(DeltaL)-expressed cells grew singly. Overexpression of TMG, but not TMG(DeltaL), decreased monolayer permeability in vitro and tumor growth in vivo. The cell-to-cell adhesion in TMG-expressed cells was Ca2+-dependent and was inhibited by monoclonal antibody against the lectin-like domain of TM. The effects of TM-mediated cell adhesion were abolished by the addition of mannose, chondroitin sulfate A, or chondroitin sulfate C. In addition, anti-lectin-like domain antibody disrupted the close clustering of the endogenous TM-expressed keratinocyte HaCaT cell line derived from normal human epidermis. Double-labeling immunofluorescence staining revealed similar distributions of TM and actin filament in the cortex region of the TMG-expressed cells. Thus, TM can function as a Ca2+-dependent cell-to-cell adhesion molecule. Binding of specific carbohydrates to the lectin-like domain is essential for this specific function.

  15. Low thermodynamic but high kinetic stability of an antifreeze protein from Rhagium mordax.

    PubMed

    Friis, Dennis S; Johnsen, Johannes L; Kristiansen, Erlend; Westh, Peter; Ramløv, Hans

    2014-06-01

    The equilibrium heat stability and the kinetic heat tolerance of a recombinant antifreeze protein (AFP) from the beetle Rhagium mordax (RmAFP1) are studied through differential scanning calorimetry and circular dichroism spectroscopy. In contrast to other insect AFPs studied with this respect, the RmAFP1 has only one disulfide bridge. The melting temperature, Tm , of the protein is determined to be 28.5°C (pH 7.4), which is much lower than most of those reported for AFPs or globular proteins in general. Despite its low melting temperature, both biophysical and activity measurements show that the protein almost completely refolds into the native state after repeated exposure of 70°C. RmAFP1 thus appears to be kinetically stable even far above its melting temperature. Thermodynamically, the insect AFPs seem to be dividable in three groups, relating to their content of disulfide bridges and widths of the ice binding motifs; high melting temperature AFPs (high disulfide content, TxT motifs), low melting temperature but high refolding capability AFPs (one disulfide bridge, TxTxTxT motifs) and irreversibly unfolded AFPs at low temperatures (no disulfide bridges, TxTxTxTxT motifs). The property of being able to cope with high temperature exposures may appear peculiar for proteins which strictly have their effect at subzero temperatures. Different aspects of this are discussed.

  16. Challenges in the expression of disulfide bonded, threonine-rich antifreeze proteins in bacteria and yeast.

    PubMed

    Tyshenko, Michael G; d'Anjou, Marc; Davies, Peter L; Daugulis, Andrew J; Walker, Virginia K

    2006-05-01

    Certain freeze-intolerant insects produce antifreeze proteins (AFPs) during overwintering including the spruce budworm (Choristoneura fumiferana) and yellow mealworm (Tenebrio molitor) AFP gene families. However, only a few of the isoforms, encoded by their multiple-copy gene families, have been characterized. When expressed in bacterial systems the insect AFPs have to be denatured and refolded in vitro, a procedure that is not uniformly successful, presumably due to the beta-helix structure and the requirement for disulfide bonds. In an attempt to overcome these difficulties, bacterial vectors and hosts that have been developed to produce soluble, folded proteins, as well as a yeast expression system (Pichia pastoris) were employed. Bacterial expression resulted in low quantities of active recombinant protein for certain isoforms. In contrast, both small and large-scale fermentation of recombinant AFP in Pichia yielded substantial protein production (100 mg/L) but functional ice binding activity of protein produced in three different transformed yeast strains (KM71, X33 or GS115) was low. Inappropriate O-linked glycosylation of the Thr-rich AFPs appeared to be partially reversed by mild chemical deglycosylation, but activity remained low. Substantial quantities, as well as activity were recovered when a fish AFP, with disulfide bonds, but without potential Thr glycosylation sites was expressed in the yeast system.

  17. Engineering a naturally inactive isoform of type III antifreeze protein into one that can stop the growth of ice.

    PubMed

    Garnham, Christopher P; Nishimiya, Yoshiyuki; Tsuda, Sakae; Davies, Peter L

    2012-11-02

    Type III antifreeze proteins (AFPs) can be sub-divided into three classes of isoforms. SP and QAE2 isoforms can slow, but not stop, the growth of ice crystals by binding to pyramidal ice planes. The other class (QAE1) binds both pyramidal and primary prism planes and is able to halt the growth of ice. Here we describe the conversion of a QAE2 isoform into a fully-active QAE1-like isoform by changing four surface-exposed residues to develop a primary prism plane binding site. Molecular dynamics analyses suggest that the basis for gain in antifreeze activity is the formation of ice-like waters on the mutated protein surface.

  18. Influence of Block Copolymerization on the Antifreeze Protein Mimetic Ice Recrystallization Inhibition Activity of Poly(vinyl alcohol).

    PubMed

    Congdon, Thomas R; Notman, Rebecca; Gibson, Matthew I

    2016-09-12

    Antifreeze (glyco) proteins are produced by many cold-acclimatized species to enable them to survive subzero temperatures. These proteins have multiple macroscopic effects on ice crystal growth which makes them appealing for low-temperature applications-from cellular cryopreservation to food storage. Poly(vinyl alcohol) has remarkable ice recrystallization inhibition activity, but its mode of action is uncertain as is the extent at which it can be incorporated into other high-order structures. Here the synthesis and characterization of well-defined block copolymers containing poly(vinyl alcohol) and poly(vinylpyrrolidone) by RAFT/MADIX polymerization is reported, as new antifreeze protein mimetics. The effect of adding a large second hydrophilic block is studied across a range of compositions, and it is found to be a passive component in ice recrystallization inhibition assays, enabling retention of all activity. In the extreme case, a block copolymer with only 10% poly(vinyl alcohol) was found to retain all activity, where statistical copolymers of PVA lose all activity with very minor changes to composition. These findings present a new method to increase the complexity of antifreeze protein mimetic materials, while retaining activity, and also to help understand the underlying mechanisms of action.

  19. Influence of Block Copolymerization on the Antifreeze Protein Mimetic Ice Recrystallization Inhibition Activity of Poly(vinyl alcohol)

    PubMed Central

    2016-01-01

    Antifreeze (glyco) proteins are produced by many cold-acclimatized species to enable them to survive subzero temperatures. These proteins have multiple macroscopic effects on ice crystal growth which makes them appealing for low-temperature applications—from cellular cryopreservation to food storage. Poly(vinyl alcohol) has remarkable ice recrystallization inhibition activity, but its mode of action is uncertain as is the extent at which it can be incorporated into other high-order structures. Here the synthesis and characterization of well-defined block copolymers containing poly(vinyl alcohol) and poly(vinylpyrrolidone) by RAFT/MADIX polymerization is reported, as new antifreeze protein mimetics. The effect of adding a large second hydrophilic block is studied across a range of compositions, and it is found to be a passive component in ice recrystallization inhibition assays, enabling retention of all activity. In the extreme case, a block copolymer with only 10% poly(vinyl alcohol) was found to retain all activity, where statistical copolymers of PVA lose all activity with very minor changes to composition. These findings present a new method to increase the complexity of antifreeze protein mimetic materials, while retaining activity, and also to help understand the underlying mechanisms of action. PMID:27476873

  20. Electro-optical properties characterization of fish type III antifreeze protein.

    PubMed

    Salvay, Andrés G; Santos, Javier; Howard, Eduardo I

    2007-12-01

    Antifreeze proteins (AFPs) are ice-binding proteins that depress the freezing point of water in a non-colligative manner without a significant modification of the melting point. Found in the blood and tissues of some organisms (such as fish, insects, plants, and soil bacteria), AFPs play an important role in subzero temperature survival. Fish Type III AFP is present in members of the subclass Zoarcoidei. AFPIII are small 7-kDa-or 14-kDa tandem-globular proteins. In the present work, we study the behavior of several physical properties, such as the low-frequency dielectric permittivity spectrum, circular dichroism, and electrical conductivity of Fish Type III AFP solutions measured at different concentrations. The combination of the information obtained from these measurements could be explained through the formation of AFP molecular aggregates or, alternatively, by the existence of some other type of interparticle interactions. Thermal stability and electro-optical behavior, when proteins are dissolved in deuterated water, were also investigated.

  1. Electro-Optical Properties Characterization of Fish Type III Antifreeze Protein

    PubMed Central

    Salvay, Andrés G.; Santos, Javier

    2008-01-01

    Antifreeze proteins (AFPs) are ice-binding proteins that depress the freezing point of water in a non-colligative manner without a significant modification of the melting point. Found in the blood and tissues of some organisms (such as fish, insects, plants, and soil bacteria), AFPs play an important role in subzero temperature survival. Fish Type III AFP is present in members of the subclass Zoarcoidei. AFPIII are small 7-kDa—or 14-kDa tandem—globular proteins. In the present work, we study the behavior of several physical properties, such as the low-frequency dielectric permittivity spectrum, circular dichroism, and electrical conductivity of Fish Type III AFP solutions measured at different concentrations. The combination of the information obtained from these measurements could be explained through the formation of AFP molecular aggregates or, alternatively, by the existence of some other type of interparticle interactions. Thermal stability and electro-optical behavior, when proteins are dissolved in deuterated water, were also investigated. PMID:19669526

  2. Differential expression of two antifreeze proteins in the desert beetle Anatolica polita (Coleoptera: Tenebriondae): seasonal variation and environmental effects.

    PubMed

    Ma, J; Wang, J; Mao, X F; Wang, Y

    2012-01-01

    Antifreeze proteins (AFPs) can inhibit and modify the growth of ice crystals. Two antifreeze protein genes, Apafp752 and Apafp914, were cloned from the desert beetle Anatolica polita (Coleoptera: Tenebriondae), and they shared 61.3 percent similarity at the amino acid level. Apafp752 also contained one variation in the most conserved TCT motif of beetle AFPs. Apafp752 and Apafp914 mRNAs had similar seasonal expression pattern. Both were stimulated by cold stress, but they expressed slightly differentially with Apafp752 being more sensitive to cold stress than Apafp914, and no more sensitive to desiccation stress than Apafp914. The thermal hysteresis activity (THA) in the beetle's hemolymph followed approximately the patterns of mRNA seasonal expression and expression upon environmental stress, with a time lag. Summer adults of the desert beetle also express mRNA of Apafp752 and Apafp914, and exhibit some hemolymph THA, suggesting other likely function of these proteins beyond antifreeze.

  3. Expression and characterization of an antifreeze protein from the perennial rye grass, Lolium perenne.

    PubMed

    Lauersen, Kyle J; Brown, Alan; Middleton, Adam; Davies, Peter L; Walker, Virginia K

    2011-06-01

    Antifreeze proteins (AFP) are an evolutionarily diverse class of stress response products best known in certain metazoans that adopt a freeze-avoidance survival strategy. The perennial ryegrass, Lolium perenne (Lp), cannot avoid winter temperatures below the crystallization point and is thought to use its LpAFP in a freeze-tolerant strategy. In order to examine properties of LpAFP in relation to L. perenne's life history, cDNA cloning, recombinant protein characterization, ice-binding activities, gene copy number, and expression responses to low temperature were examined. Transcripts, encoded by only a few gene copies, appeared to increase in abundance after diploid plants were transferred to 4°C for 1-2 days, and in parallel with the ice recrystallization inhibition activities. Circular dichroism spectra of recombinant LpAFP showed three clear folding transition temperatures including one between 10 and 15°C, suggesting to us that folding modifications of the secreted AFP could allow the targeted degradation of the protein in planta when temperatures increase. Although LpAFP showed low thermal hysteresis activity and partitioning into ice, it was similar to AFPs from freeze-avoiding organisms in other respects. Therefore, the type of low temperature resistance strategy adopted by a particular species may not depend on the type of AFP. The independence of AFP sequence and life-history has practical implications for the development of genetically-modified crops with enhanced freeze tolerance.

  4. Isolation and characterization of type I antifreeze proteins from cunner, Tautogolabrus adspersus, order Perciformes.

    PubMed

    Hobbs, Rod S; Shears, Margaret A; Graham, Laurie A; Davies, Peter L; Fletcher, Garth L

    2011-10-01

    Antifreeze proteins (AFPs) are produced by many species of teleost fish that inhabit potentially lethal ice-laden seawater and afford them protection from freezing. To date type I AFPs have been fully characterized in two teleost orders: Pleuronectiformes and Scorpaeniformes. In this study, we report the isolation and complete characterization of a type I AFP present in fish from a third order: cunner (Tautogolabrus adspersus), order Perciformes (family Labridae). This protein was purified from blood plasma and found to belong to what is now known as classical type I AFP with their small size (mass 4095.16 Da), alanine richness (> 57 mol%), high α-helicity (> 99%) with the ability to undergo reversible thermal denaturation, 11 amino acid (ThrX(10)) repeat regions within the primary structure, the capacity to impart a hexagonal bipyramidal shaping to ice crystals and the conservation of an ice-binding site found in many of the other type I AFPs. Partial de novo sequencing of the plasma AFP accounted for approximately half of the peptide mass. Sequencing of a combined liver and skin cDNA library indicated that the protein is produced without a signal sequence. In addition the translated product of the AFP cDNA suggests that it codes for the AFP isolated from plasma. These results further solidify the hypothesis that type I AFPs are multiphyletic in origin and suggest that they represent remarkable examples of convergent evolution within three orders of teleost fish.

  5. Antifreeze (glyco)protein mimetic behavior of poly(vinyl alcohol): detailed structure ice recrystallization inhibition activity study.

    PubMed

    Congdon, Thomas; Notman, Rebecca; Gibson, Matthew I

    2013-05-13

    This manuscript reports a detailed study on the ability of poly(vinyl alcohol) to act as a biomimetic surrogate for antifreeze(glyco)proteins, with a focus on the specific property of ice-recrystallization inhibition (IRI). Despite over 40 years of study, the underlying mechanisms that govern the action of biological antifreezes are still poorly understood, which is in part due to their limited availability and challenging synthesis. Poly(vinyl alcohol) (PVA) has been shown to display remarkable ice recrystallization inhibition activity despite its major structural differences to native antifreeze proteins. Here, controlled radical polymerization is used to synthesize well-defined PVA, which has enabled us to obtain the first quantitative structure-activity relationships, to probe the role of molecular weight and comonomers on IRI activity. Crucially, it was found that IRI activity is "switched on" when the polymer chain length increases from 10 and 20 repeat units. Substitution of the polymer side chains with hydrophilic or hydrophobic units was found to diminish activity. Hydrophobic modifications to the backbone were slightly more tolerated than side chain modifications, which implies an unbroken sequence of hydroxyl units is necessary for activity. These results highlight that, although hydrophobic domains are key components of IRI activity, the random inclusion of addition hydrophobic units does not guarantee an increase in activity and that the actual polymer conformation is important.

  6. Structural Basis for the Inhibition of Gas Hydrates by α-Helical Antifreeze Proteins.

    PubMed

    Sun, Tianjun; Davies, Peter L; Walker, Virginia K

    2015-10-20

    Kinetic hydrate inhibitors (KHIs) are used commercially to inhibit gas hydrate formation and growth in pipelines. However, improvement of these polymers has been constrained by the lack of verified molecular models. Since antifreeze proteins (AFPs) act as KHIs, we have used their solved x-ray crystallographic structures in molecular modeling to explore gas hydrate inhibition. The internal clathrate water network of the fish AFP Maxi, which extends to the protein's outer surface, is remarkably similar to the {100} planes of structure type II (sII) gas hydrate. The crystal structure of this water web has facilitated the construction of in silico models for Maxi and type I AFP binding to sII hydrates. Here, we have substantiated our models with experimental evidence of Maxi binding to the tetrahydrofuran sII model hydrate. Both in silico and experimental evidence support the absorbance-inhibition mechanism proposed for KHI binding to gas hydrates. Based on the Maxi crystal structure we suggest that the inhibitor adsorbs to the gas hydrate lattice through the same anchored clathrate water mechanism used to bind ice. These results will facilitate the rational design of a next generation of effective green KHIs for the petroleum industry to ensure safe and efficient hydrocarbon flow.

  7. Dendrimer-Linked Antifreeze Proteins Have Superior Activity and Thermal Recovery.

    PubMed

    Stevens, Corey A; Drori, Ran; Zalis, Shiran; Braslavsky, Ido; Davies, Peter L

    2015-09-16

    By binding to ice, antifreeze proteins (AFPs) depress the freezing point of a solution and inhibit ice recrystallization if freezing does occur. Previous work showed that the activity of an AFP was incrementally increased by fusing it to another protein. Even larger increases in activity were achieved by doubling the number of ice-binding sites by dimerization. Here, we have combined the two strategies by linking multiple outward-facing AFPs to a dendrimer to significantly increase both the size of the molecule and the number of ice-binding sites. Using a heterobifunctional cross-linker, we attached between 6 and 11 type III AFPs to a second-generation polyamidoamine (G2-PAMAM) dendrimer with 16 reactive termini. This heterogeneous sample of dendrimer-linked type III constructs showed a greater than 4-fold increase in freezing point depression over that of monomeric type III AFP. This multimerized AFP was particularly effective at ice recrystallization inhibition activity, likely because it can simultaneously bind multiple ice surfaces. Additionally, attachment to the dendrimer has afforded the AFP superior recovery from heat denaturation. Linking AFPs together via polymers can generate novel reagents for controlling ice growth and recrystallization.

  8. Perturbation of bacterial ice nucleation activity by a grass antifreeze protein.

    PubMed

    Tomalty, Heather E; Walker, Virginia K

    2014-09-26

    Certain plant-associating bacteria produce ice nucleation proteins (INPs) which allow the crystallization of water at high subzero temperatures. Many of these microbes are considered plant pathogens since the formed ice can damage tissues, allowing access to nutrients. Intriguingly, certain plants that host these bacteria synthesize antifreeze proteins (AFPs). Once freezing has occurred, plant AFPs likely function to inhibit the growth of large damaging ice crystals. However, we postulated that such AFPs might also serve as defensive mechanisms against bacterial-mediated ice nucleation. Recombinant AFP derived from the perennial ryegrass Lolium perenne (LpAFP) was combined with INP preparations originating from the grass epiphyte, Pseudomonas syringae. The presence of INPs had no effect on AFP activity, including thermal hysteresis and ice recrystallization inhibition. Strikingly, the ice nucleation point of the INP was depressed up to 1.9°C in the presence of LpAFP, but a recombinant fish AFP did not lower the INP-imposed freezing point. Assays with mutant LpAFPs and the visualization of bacterially-displayed fluorescent plant AFP suggest that INP and LpAFP can interact. Thus, we postulate that in addition to controlling ice growth, plant AFPs may also function as a defensive strategy against the damaging effects of ice-nucleating bacteria.

  9. Vimentin and desmin possess GlcNAc-binding lectin-like properties on cell surfaces.

    PubMed

    Ise, Hirohiko; Kobayashi, Satoshi; Goto, Mitsuaki; Sato, Takao; Kawakubo, Masatomo; Takahashi, Masafumi; Ikeda, Uichi; Akaike, Toshihiro

    2010-07-01

    Vimentin and desmin are intermediate filament proteins found in various mesenchymal and skeletal muscle cells, respectively. These proteins play an important role in the stabilization of the cytoplasmic architecture. Here, we found, using artificial biomimicking glycopolymers, that vimentin and desmin possess N-acetylglucosamine (GlcNAc)-binding lectin-like properties on the cell surfaces of various vimentin- and desmin-expressing cells such as cardiomyocytes and vascular smooth muscle cells. The rod II domain of these proteins was demonstrated to be localized to the cell surface and to directly bind to the artificial biomimicking GlcNAc-bearing polymer, by confocal laser microscopy and surface plasmon resonance analysis. These glycopolymers strongly interact with lectins and are useful tools for the analysis of lectin-carbohydrate interactions, since glycopolymers binding to lectins can induce the clustering of lectins due to multivalent glycoside ligand binding. Moreover, immunocytochemistry and pull-down assay with His-tagged vimentin-rod II domain protein showed that the vimentin-rod II domain interacts with O-GlcNAc proteins. These results suggest that O-GlcNAc proteins might be one candidate for physiological GlcNAc-bearing ligands with which vimentin and desmin interact. These findings demonstrate a novel function of vimentin and desmin that does not involve stabilization of the cytoplasmic architecture by which these proteins interact with physiological GlcNAc-bearing ligands such as O-GlcNAc proteins on the cell surface through their GlcNAc-binding lectin-like properties.

  10. Determining the Ice-binding Planes of Antifreeze Proteins by Fluorescence-based Ice Plane Affinity

    PubMed Central

    Basu, Koli; Garnham, Christopher P.; Nishimiya, Yoshiyuki; Tsuda, Sakae; Braslavsky, Ido; Davies, Peter

    2014-01-01

    Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms. PMID:24457629

  11. Investigation of changes in structure and thermodynamic of spruce budworm antifreeze protein under subfreezing temperature

    NASA Astrophysics Data System (ADS)

    Nguyen, Hung; Le, Ly

    2017-01-01

    The aim of this theoretical work is to investigate of the changes in structure and thermodynamics of spruce budworm antifreeze protein (sbAFP) at low temperatures by using molecular dynamics simulation. The aqueous solution will form ice crystal network under the vaguely hexagonal shape at low temperature and fully represented the characteristics of hydrophobic interaction. Like ice crystal network, the cyclohexane region (including cyclohexane molecules) have enough of the characteristics of hydrophobic interaction. Therefore, in this research the cyclohexane region will be used as a representation of ice crystal network to investigate the interactions of sbAFP and ice crystal network at low temperature. The activity of sbAFP in subfreezing environment, therefore, can be clearly observed via the changes of the hydrophobic (cyclohexane region) and hydrophilic (water region) interactions. The obtained results from total energies, hydrogen bond lifetime correlation C(t), radial distribution function, mean square deviation and snapshots of sbAFP complexes indicated that sbAFP has some special changes in structure and interaction with water and cyclohexane regions at 278 K, as being transition temperature point of water molecules in sbAFP complex at low temperatures, which is more structured and support the experimental observation that the sbAFP complex becomes more rigid as the temperature is lowered.

  12. Structural Basis for the Inhibition of Gas Hydrates by α-Helical Antifreeze Proteins

    PubMed Central

    Sun, Tianjun; Davies, Peter L.; Walker, Virginia K.

    2015-01-01

    Kinetic hydrate inhibitors (KHIs) are used commercially to inhibit gas hydrate formation and growth in pipelines. However, improvement of these polymers has been constrained by the lack of verified molecular models. Since antifreeze proteins (AFPs) act as KHIs, we have used their solved x-ray crystallographic structures in molecular modeling to explore gas hydrate inhibition. The internal clathrate water network of the fish AFP Maxi, which extends to the protein’s outer surface, is remarkably similar to the {100} planes of structure type II (sII) gas hydrate. The crystal structure of this water web has facilitated the construction of in silico models for Maxi and type I AFP binding to sII hydrates. Here, we have substantiated our models with experimental evidence of Maxi binding to the tetrahydrofuran sII model hydrate. Both in silico and experimental evidence support the absorbance-inhibition mechanism proposed for KHI binding to gas hydrates. Based on the Maxi crystal structure we suggest that the inhibitor adsorbs to the gas hydrate lattice through the same anchored clathrate water mechanism used to bind ice. These results will facilitate the rational design of a next generation of effective green KHIs for the petroleum industry to ensure safe and efficient hydrocarbon flow. PMID:26488661

  13. A mechanism for stabilization of membranes at low temperatures by an antifreeze protein.

    PubMed Central

    Tomczak, Melanie M; Hincha, Dirk K; Estrada, Sergio D; Wolkers, Willem F; Crowe, Lois M; Feeney, Robert E; Tablin, Fern; Crowe, John H

    2002-01-01

    Polar fish, cold hardy plants, and overwintering insects produce antifreeze proteins (AFPs), which lower the freezing point of solutions noncolligatively and inhibit ice crystal growth. Fish AFPs have been shown to stabilize membranes and cells in vitro during hypothermic storage, probably by interacting with the plasma membrane, but the mechanism of this stabilization has not been clear. We show here that during chilling to nonfreezing temperatures the alpha-helical AFP type I from polar fish inhibits leakage across model membranes containing an unsaturated chloroplast galactolipid. The mechanism involves binding of the AFP to the bilayer, which increases the phase transition temperature of the membranes and alters the molecular packing of the acyl chains. We suggest that this change in acyl chain packing results in the reduced membrane permeability. The data suggest a hydrophobic interaction between the peptide and the bilayer. Further, we suggest that the expression of AFP type I in transgenic plants may be significant for thermal adaptation of chilling-sensitive plants. PMID:11806929

  14. Investigation of changes in structure and thermodynamic of spruce budworm antifreeze protein under subfreezing temperature

    PubMed Central

    Nguyen, Hung; Le, Ly

    2017-01-01

    The aim of this theoretical work is to investigate of the changes in structure and thermodynamics of spruce budworm antifreeze protein (sbAFP) at low temperatures by using molecular dynamics simulation. The aqueous solution will form ice crystal network under the vaguely hexagonal shape at low temperature and fully represented the characteristics of hydrophobic interaction. Like ice crystal network, the cyclohexane region (including cyclohexane molecules) have enough of the characteristics of hydrophobic interaction. Therefore, in this research the cyclohexane region will be used as a representation of ice crystal network to investigate the interactions of sbAFP and ice crystal network at low temperature. The activity of sbAFP in subfreezing environment, therefore, can be clearly observed via the changes of the hydrophobic (cyclohexane region) and hydrophilic (water region) interactions. The obtained results from total energies, hydrogen bond lifetime correlation C(t), radial distribution function, mean square deviation and snapshots of sbAFP complexes indicated that sbAFP has some special changes in structure and interaction with water and cyclohexane regions at 278 K, as being transition temperature point of water molecules in sbAFP complex at low temperatures, which is more structured and support the experimental observation that the sbAFP complex becomes more rigid as the temperature is lowered. PMID:28106056

  15. New insights into ice growth and melting modifications by antifreeze proteins

    PubMed Central

    Bar-Dolev, Maya; Celik, Yeliz; Wettlaufer, J. S.; Davies, Peter L.; Braslavsky, Ido

    2012-01-01

    Antifreeze proteins (AFPs) evolved in many organisms, allowing them to survive in cold climates by controlling ice crystal growth. The specific interactions of AFPs with ice determine their potential applications in agriculture, food preservation and medicine. AFPs control the shapes of ice crystals in a manner characteristic of the particular AFP type. Moderately active AFPs cause the formation of elongated bipyramidal crystals, often with seemingly defined facets, while hyperactive AFPs produce more varied crystal shapes. These different morphologies are generally considered to be growth shapes. In a series of bright light and fluorescent microscopy observations of ice crystals in solutions containing different AFPs, we show that crystal shaping also occurs during melting. In particular, the characteristic ice shapes observed in solutions of most hyperactive AFPs are formed during melting. We relate these findings to the affinities of the hyperactive AFPs for the basal plane of ice. Our results demonstrate the relation between basal plane affinity and hyperactivity and show a clear difference in the ice-shaping mechanisms of most moderate and hyperactive AFPs. This study provides key aspects associated with the identification of hyperactive AFPs. PMID:22787007

  16. New insights into ice growth and melting modifications by antifreeze proteins.

    PubMed

    Bar-Dolev, Maya; Celik, Yeliz; Wettlaufer, J S; Davies, Peter L; Braslavsky, Ido

    2012-12-07

    Antifreeze proteins (AFPs) evolved in many organisms, allowing them to survive in cold climates by controlling ice crystal growth. The specific interactions of AFPs with ice determine their potential applications in agriculture, food preservation and medicine. AFPs control the shapes of ice crystals in a manner characteristic of the particular AFP type. Moderately active AFPs cause the formation of elongated bipyramidal crystals, often with seemingly defined facets, while hyperactive AFPs produce more varied crystal shapes. These different morphologies are generally considered to be growth shapes. In a series of bright light and fluorescent microscopy observations of ice crystals in solutions containing different AFPs, we show that crystal shaping also occurs during melting. In particular, the characteristic ice shapes observed in solutions of most hyperactive AFPs are formed during melting. We relate these findings to the affinities of the hyperactive AFPs for the basal plane of ice. Our results demonstrate the relation between basal plane affinity and hyperactivity and show a clear difference in the ice-shaping mechanisms of most moderate and hyperactive AFPs. This study provides key aspects associated with the identification of hyperactive AFPs.

  17. Ice restructuring inhibition activities in antifreeze proteins with distinct differences in thermal hysteresis.

    PubMed

    Yu, Sally O; Brown, Alan; Middleton, Adam J; Tomczak, Melanie M; Walker, Virginia K; Davies, Peter L

    2010-12-01

    Antifreeze proteins (AFPs) share two related properties: the ability to depress the freezing temperature below the melting point of ice (thermal hysteresis; TH); and the ability to inhibit the restructuring of ice into larger crystals. Since the 'hyperactive' AFPs, which have been more recently discovered, show an order of magnitude more TH than previously characterized AFPs, we have now determined their activities in ice restructuring inhibition (IrI) assays. IrI activities of three TH-hyperactive AFPs and three less TH-active AFPs varied over an 8-fold range. There was no obvious correlation between high TH activity and high IrI activity. However, the use of mutant AFPs demonstrated that severe disruption of ice-binding residues diminished both TH and IrI similarly, revealing that that the same ice-binding residues are crucial for both activities. In addition, bicarbonate ions, which are known to enhance the TH activity of AFPs, also enhanced their IrI activity. We suggest that these seemingly contradictory observations can be partially explained by differences in the coverage of ice by TH-hyperactive and non-hyperactive AFPs, and by differences in the stability of AFP-bound ice under supercooled and recrystallization conditions.

  18. Comparative modeling of the three-dimensional structure of type II antifreeze protein.

    PubMed Central

    Sönnichsen, F. D.; Sykes, B. D.; Davies, P. L.

    1995-01-01

    Type II antifreeze proteins (AFP), which inhibit the growth of seed ice crystals in the blood of certain fishes (sea raven, herring, and smelt), are the largest known fish AFPs and the only class for which detailed structural information is not yet available. However, a sequence homology has been recognized between these proteins and the carbohydrate recognition domain of C-type lectins. The structure of this domain from rat mannose-binding protein (MBP-A) has been solved by X-ray crystallography (Weis WI, Drickamer K, Hendrickson WA, 1992, Nature 360:127-134) and provided the coordinates for constructing the three-dimensional model of the 129-amino acid Type II AFP from sea raven, to which it shows 19% sequence identity. Multiple sequence alignments between Type II AFPs, pancreatic stone protein, MBP-A, and as many as 50 carbohydrate-recognition domain sequences from various lectins were performed to determine reliably aligned sequence regions. Successive molecular dynamics and energy minimization calculations were used to relax bond lengths and angles and to identify flexible regions. The derived structure contains two alpha-helices, two beta-sheets, and a high proportion of amino acids in loops and turns. The model is in good agreement with preliminary NMR spectroscopic analyses. It explains the observed differences in calcium binding between sea raven Type II AFP and MBP-A. Furthermore, the model proposes the formation of five disulfide bridges between Cys 7 and Cys 18, Cys 35 and Cys 125, Cys 69 and Cys 100, Cys 89 and Cys 111, and Cys 101 and Cys 117.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7540906

  19. Growth inhibition at the ice prismatic plane induced by a spruce budworm antifreeze protein: a molecular dynamics simulation study.

    PubMed

    Nada, H; Furukawa, Y

    2011-11-28

    A molecular dynamics simulation was conducted to investigate the growth kinetics at the ice prismatic interface to which a spruce budworm antifreeze protein was bound. Two initial binding conformations of the protein at the interface--one energetically stable and the other energetically unstable--were examined. For both binding conformations, the growth of ice was observed around the protein. A sharp decrease in the rate of ice growth was observed around the protein that initially had the energetically stable binding conformation. Simulation results suggest that the observed decrease in the ice growth rate was attributable to melting point depression caused by the Gibbs-Thomson effect. The protein that initially had the energetically unstable binding conformation markedly relaxed so as to stably bind to the prismatic plane interface of the grown ice; thereafter, a decrease in the ice growth rate was observed as well. However, the binding conformation that the protein approached during the relaxation was different from that of the protein that initially had the energetically stable binding conformation. Thus, the simulation indicates the existence of two binding conformations for inducing a decrease in the ice growth rate. The results are possibly related to the hyperactivity of a spruce budworm antifreeze protein in real systems.

  20. X-ray Structure of Snow Flea Antifreeze Protein Determined by Racemic Crystallization of Synthetic Protein Enantiomers

    SciTech Connect

    Pentelute, Brad L.; Gates, Zachary P.; Tereshko, Valentina; Dashnau, Jennifer L.; Vanderkooi, Jane M.; Kossiakoff, Anthony A.; Kent, Stephen B.H.

    2008-08-20

    Chemical protein synthesis and racemic protein crystallization were used to determine the X-ray structure of the snow flea antifreeze protein (sfAFP). Crystal formation from a racemic solution containing equal amounts of the chemically synthesized proteins d-sfAFP and l-sfAFP occurred much more readily than for l-sfAFP alone. More facile crystal formation also occurred from a quasi-racemic mixture of d-sfAFP and l-Se-sfAFP, a chemical protein analogue that contains an additional -SeCH2- moiety at one residue and thus differs slightly from the true enantiomer. Multiple wavelength anomalous dispersion (MAD) phasing from quasi-racemate crystals was then used to determine the X-ray structure of the sfAFP protein molecule. The resulting model was used to solve by molecular replacement the X-ray structure of l-sfAFP to a resolution of 0.98 {angstrom}. The l-sfAFP molecule is made up of six antiparallel left-handed PPII helixes, stacked in two sets of three, to form a compact brick-like structure with one hydrophilic face and one hydrophobic face. This is a novel experimental protein structure and closely resembles a structural model proposed for sfAFP. These results illustrate the utility of total chemical synthesis combined with racemic crystallization and X-ray crystallography for determining the unknown structure of a protein.

  1. Neutron structure of type-III antifreeze protein allows the reconstruction of AFP-ice interface.

    PubMed

    Howard, Eduardo I; Blakeley, Matthew P; Haertlein, Michael; Petit-Haertlein, Isabelle; Mitschler, Andre; Fisher, Stuart J; Cousido-Siah, Alexandra; Salvay, Andrés G; Popov, Alexandre; Muller-Dieckmann, Christoph; Petrova, Tatiana; Podjarny, Alberto

    2011-01-01

    Antifreeze proteins (AFPs) inhibit ice growth at sub-zero temperatures. The prototypical type-III AFPs have been extensively studied, notably by X-ray crystallography, solid-state and solution NMR, and mutagenesis, leading to the identification of a compound ice-binding surface (IBS) composed of two adjacent ice-binding sections, each which binds to particular lattice planes of ice crystals, poisoning their growth. This surface, including many hydrophobic and some hydrophilic residues, has been extensively used to model the interaction of AFP with ice. Experimentally observed water molecules facing the IBS have been used in an attempt to validate these models. However, these trials have been hindered by the limited capability of X-ray crystallography to reliably identify all water molecules of the hydration layer. Due to the strong diffraction signal from both the oxygen and deuterium atoms, neutron diffraction provides a more effective way to determine the water molecule positions (as D(2) O). Here we report the successful structure determination at 293 K of fully perdeuterated type-III AFP by joint X-ray and neutron diffraction providing a very detailed description of the protein and its solvent structure. X-ray data were collected to a resolution of 1.05 Å, and neutron Laue data to a resolution of 1.85 Å with a "radically small" crystal volume of 0.13 mm(3). The identification of a tetrahedral water cluster in nuclear scattering density maps has allowed the reconstruction of the IBS-bound ice crystal primary prismatic face. Analysis of the interactions between the IBS and the bound ice crystal primary prismatic face indicates the role of the hydrophobic residues, which are found to bind inside the holes of the ice surface, thus explaining the specificity of AFPs for ice versus water.

  2. Multivalent Display of Antifreeze Proteins by Fusion to Self-Assembling Protein Cages Enhances Ice-Binding Activities.

    PubMed

    Phippen, Sean W; Stevens, Corey A; Vance, Tyler D R; King, Neil P; Baker, David; Davies, Peter L

    2016-12-13

    Antifreeze proteins (AFPs) are small monomeric proteins that adsorb to the surface of ice to inhibit ice crystal growth and impart freeze resistance to the organisms producing them. Previously, monomeric AFPs have been conjugated to the termini of branched polymers to increase their activity through the simultaneous binding of more than one AFP to ice. Here, we describe a superior approach to increasing AFP activity through oligomerization that eliminates the need for conjugation reactions with varying levels of efficiency. A moderately active AFP from a fish and a hyperactive AFP from an Antarctic bacterium were genetically fused to the C-termini of one component of the 24-subunit protein cage T33-21, resulting in protein nanoparticles that multivalently display exactly 12 AFPs. The resulting nanoparticles exhibited freezing point depression >50-fold greater than that seen with the same concentration of monomeric AFP and a similar increase in the level of ice-recrystallization inhibition. These results support the anchored clathrate mechanism of binding of AFP to ice. The enhanced freezing point depression could be due to the difficulty of overgrowing a larger AFP on the ice surface and the improved ice-recrystallization inhibition to the ability of the nanoparticle to simultaneously bind multiple ice grains. Oligomerization of these proteins using self-assembling protein cages will be useful in a variety of biotechnology and cryobiology applications.

  3. Lectin-Like Molecules of Lactobacillus rhamnosus GG Inhibit Pathogenic Escherichia coli and Salmonella Biofilm Formation

    PubMed Central

    Petrova, Mariya I.; Imholz, Nicole C. E.; Verhoeven, Tine L. A.; Balzarini, Jan; Van Damme, Els J. M.; Schols, Dominique; Vanderleyden, Jos; Lebeer, Sarah

    2016-01-01

    Objectives Increased antibiotic resistance has catalyzed the research on new antibacterial molecules and alternative strategies, such as the application of beneficial bacteria. Since lectin molecules have unique sugar-recognizing capacities, and pathogens are often decorated with sugars that affect their survival and infectivity, we explored whether lectins from the probiotic strain Lactobacillus rhamnosus GG have antipathogenic properties. Methods The genome sequence of L. rhamnosus GG was screened for the presence of lectin-like proteins. Two genes, LGG_RS02780 and LGG_RS02750, encoding for polypeptides with an N-terminal conserved L-type lectin domain were detected and designated Llp1 (lectin-like protein 1) and Llp2. The capacity of Llp1 and Llp2 to inhibit biofilm formation of various pathogens was investigated. Sugar specificity was determined by Sepharose beads assays and glycan array screening. Results The isolated lectin domains of Llp1 and Llp2 possess pronounced inhibitory activity against biofilm formation by various pathogens, including clinical Salmonella species and uropathogenic E. coli, with Llp2 being more active than Llp1. In addition, sugar binding assays with Llp1 and Llp2 indicate specificity for complex glycans. Both proteins are also involved in the adhesion capacity of L. rhamnosus GG to gastrointestinal and vaginal epithelial cells. Conclusions Lectins isolated from or expressed by beneficial lactobacilli could be considered promising bio-active ingredients for improved prophylaxis of urogenital and gastrointestinal infections. PMID:27537843

  4. Hydrophobic tendency of polar group hydration as a major force in type I antifreeze protein recognition.

    PubMed

    Yang, Cheng; Sharp, Kim A

    2005-05-01

    The random network model of water quantitatively describes the different hydration heat capacities of polar and apolar solutes in terms of distortions of the water-water hydrogen bonding angle in the first hydration shell (Gallagher and Sharp, JACS 2003;125:9853). The distribution of this angle in pure water is bimodal, with a low-angle population and high-angle population. Polar solutes increase the high-angle population while apolar solutes increase the low-angle population. The ratio of the two populations quantifies the hydrophobicity of the solute and provides a sensitive measure of water structural distortions. This method of analysis is applied to study hydration of type I thermal hysteresis protein (THP) from winter flounder and three quadruple mutants of four threonine residues at positions 2, 13, 24, and 35. Wild-type and two mutants (VVVV and AAAA) have antifreeze (thermal hysteresis) activity, while the other mutant (SSSS) has no activity. The analysis reveals significant differences in the hydration structure of the ice-binding site. For the SSSS mutant, polar groups have a typical polar-like hydration, that is, more high-angle H-bonds than bulk water. For the wild-type and active mutants, polar groups have unusual, very apolar-like hydration, that is, more low-angle H-bonds than bulk water. This pattern of hydration was seen previously in the structurally distinct type III THPs (Yang & Sharp Biophys Chem 2004;109:137), suggesting for the first time a general mechanism for different THP classes. The specific shape, residue size, and clustering of both polar and apoler groups are essential for an active ice binding surface.

  5. Recombinant Dendroides canadensis antifreeze proteins as potential ingredients in cryopreservation solutions

    PubMed Central

    Halwani, Dina O.; Brockbank, Kelvin G.M.; Duman, John G.; Campbell, Lia H.

    2015-01-01

    Expanding cryopreservation methods to include a wider range of cell types, such as those sensitive to freezing, is needed for maintaining the viability of cell-based regenerative medicine products. Conventional cryopreservation protocols, which include use of cryoprotectants such as dimethylsulfoxide (Me2SO), have not prevented ice-induced damage to cell and tissue matrices during freezing. A family of antifreeze proteins (AFPs) produced in the larvae of the beetle, Dendroides canadensis allow this insect to survive subzero temperatures as low as −26°C. This study is an assessment of the effect of the four hemolymph D. canadensis AFPs (DAFPs) on the supercooling (nucleating) temperature, ice structure patterns and viability of the A10 cell line derived from the thoracic aorta of embryonic rat. Cryoprotectant solution cocktails containing combinations of DAFPs in concentrations ranging from 0–3mg/mL in Unisol base mixed with 1M Me2SO were first evaluated by cryomicroscopy. Combining multiple DAFPs demonstrated significant supercooling point depressing activity (~9°C) when compared to single DAFPs and/or conventional 1M Me2SO control solutions. Concentrations of DAFPs as low as 1μg/mL were sufficient to trigger this effect. In addition, significantly improved A10 smooth muscle cell viability was observed in cryopreservation experiments with low DAFP-6 and DAFP-2 concentrations in combination with Me2SO. No significant improvement in viability was observed with either DAFP-1 or DAFP-4. Low and effective DAFP concentrations are advantageous because they minimize concerns regarding cell cytotoxicity and manufacturing cost. These findings support the potential of incorporating DAFPs in solutions used to cryopreserve cells and tissues. PMID:24662031

  6. A family of expressed antifreeze protein genes from the moth, Choristoneura fumiferana.

    PubMed

    Doucet, Daniel; Tyshenko, Michael G; Davies, Peter L; Walker, Virginia K

    2002-01-01

    The freeze-intolerant insect, Choristoneura fumiferana (spruce budworm), produces multiple antifreeze protein (AFP) isoforms for protection during the overwintering stage. We now report the cloning of AFP genes from insects; Afp-Lu1 encodes a approximately 9-kDa AFP isoform, and Afp-Iu1 encodes a approximately 12-kDa AFP isoform. Both CfAFP genes have similar structures with a single 3- to 3.6-kb intron interrupting the coding region. The second exon of an additional CfAFP gene, 2.7a, encoding a new approximately 9-kDa isoform, was found 3.7 kb upstream of Afp-Lu1 and demonstrates that some AFP family members are linked in tandem. This gene appears to encode an AFP with 68-76% identity to previously isolated CfAFPs. With its eight Cys residues necessary for disulfide bonding and five perfectly conserved 'Thr button' (Thr-Xaa-Thr) ice-binding motifs, it can be modeled as a functional AFP. Southern blot analysis shows that there are approximately 17 genes in this AFP family, with each of the isoforms represented by two to five gene copies. Transcript accumulation from Afp-Lu1 and Afp-Iu1 (or closely related genes) was maximal during the overwintering stage, while 2.7a transcripts were only detected in first instars, larvae that are normally found only in the summer. Contrary to expectations, this differential expression demonstrates that CfAFP gene family transcripts are primarily regulated during development, rather than by seasonally low temperatures.

  7. Antifreeze protein-induced superheating of ice inside Antarctic notothenioid fishes inhibits melting during summer warming.

    PubMed

    Cziko, Paul A; DeVries, Arthur L; Evans, Clive W; Cheng, Chi-Hing Christina

    2014-10-07

    Antifreeze proteins (AFPs) of polar marine teleost fishes are widely recognized as an evolutionary innovation of vast adaptive value in that, by adsorbing to and inhibiting the growth of internalized environmental ice crystals, they prevent death by inoculative freezing. Paradoxically, systemic accumulation of AFP-stabilized ice could also be lethal. Whether or how fishes eliminate internal ice is unknown. To investigate if ice inside high-latitude Antarctic notothenioid fishes could melt seasonally, we measured its melting point and obtained a decadal temperature record from a shallow benthic fish habitat in McMurdo Sound, Antarctica. We found that AFP-stabilized ice resists melting at temperatures above the expected equilibrium freezing/melting point (eqFMP), both in vitro and in vivo. Superheated ice was directly observed in notothenioid serum samples and in solutions of purified AFPs, and ice was found to persist inside live fishes at temperatures more than 1 °C above their eqFMP for at least 24 h, and at a lower temperature for at least several days. Field experiments confirmed that superheated ice occurs naturally inside wild fishes. Over the long-term record (1999-2012), seawater temperature surpassed the fish eqFMP in most summers, but never exceeded the highest temperature at which ice persisted inside experimental fishes. Thus, because of the effects of AFP-induced melting inhibition, summer warming may not reliably eliminate internal ice. Our results expose a potentially antagonistic pleiotropic effect of AFPs: beneficial freezing avoidance is accompanied by melting inhibition that may contribute to lifelong accumulation of detrimental internal ice crystals.

  8. Comparative Proteome Analysis of Cryopreserved Flagella and Head Plasma Membrane Proteins from Sea Bream Spermatozoa: Effect of Antifreeze Proteins

    PubMed Central

    Zilli, Loredana; Beirão, José; Schiavone, Roberta; Herraez, Maria Paz; Gnoni, Antonio; Vilella, Sebastiano

    2014-01-01

    Cryopreservation induces injuries to fish spermatozoa that in turn affect sperm quality in terms of fertilization ability, motility, DNA and protein integrity and larval survival. To reduce the loss of sperm quality due to freezing-thawing, it is necessary to improve these procedures. In the present study we investigated the ability of two antifreeze proteins (AFPI and AFPIII) to reduce the loss of quality of sea bream spermatozoa due to cryopreservation. To do so, we compared viability, motility, straight-line velocity and curvilinear velocity of fresh and (AFPs)-cryopreserved spermatozoa. AFPIII addition to cryopreservation medium improved viability, motility and straight-line velocity with respect to DMSO or DMSO plus AFPI. To clarify the molecular mechanism(s) underlying these findings, the protein profile of two different cryopreserved sperm domains, flagella and head plasma membranes, was analysed. The protein profiles differed between fresh and frozen-thawed semen and results of the image analysis demonstrated that, after cryopreservation, out of 270 proteins 12 were decreased and 7 were increased in isolated flagella, and out of 150 proteins 6 showed a significant decrease and 4 showed a significant increase in head membranes. Mass spectrometry analysis identified 6 proteins (4 from isolated flagella and 2 present both in flagella and head plasma membranes) within the protein spots affected by the freezing-thawing procedure. 3 out of 4 proteins from isolated flagella were involved in the sperm bioenergetic system. Our results indicate that the ability of AFPIII to protect sea bream sperm quality can be, at least in part, ascribed to reducing changes in the sperm protein profile occurring during the freezing-thawing procedure. Our results clearly demonstrated that AFPIII addition to cryopreservation medium improved the protection against freezing respect to DMSO or DMSO plus AFPI. In addition we propose specific proteins of spermatozoa as markers related to

  9. Comparative proteome analysis of cryopreserved flagella and head plasma membrane proteins from sea bream spermatozoa: effect of antifreeze proteins.

    PubMed

    Zilli, Loredana; Beirão, José; Schiavone, Roberta; Herraez, Maria Paz; Gnoni, Antonio; Vilella, Sebastiano

    2014-01-01

    Cryopreservation induces injuries to fish spermatozoa that in turn affect sperm quality in terms of fertilization ability, motility, DNA and protein integrity and larval survival. To reduce the loss of sperm quality due to freezing-thawing, it is necessary to improve these procedures. In the present study we investigated the ability of two antifreeze proteins (AFPI and AFPIII) to reduce the loss of quality of sea bream spermatozoa due to cryopreservation. To do so, we compared viability, motility, straight-line velocity and curvilinear velocity of fresh and (AFPs)-cryopreserved spermatozoa. AFPIII addition to cryopreservation medium improved viability, motility and straight-line velocity with respect to DMSO or DMSO plus AFPI. To clarify the molecular mechanism(s) underlying these findings, the protein profile of two different cryopreserved sperm domains, flagella and head plasma membranes, was analysed. The protein profiles differed between fresh and frozen-thawed semen and results of the image analysis demonstrated that, after cryopreservation, out of 270 proteins 12 were decreased and 7 were increased in isolated flagella, and out of 150 proteins 6 showed a significant decrease and 4 showed a significant increase in head membranes. Mass spectrometry analysis identified 6 proteins (4 from isolated flagella and 2 present both in flagella and head plasma membranes) within the protein spots affected by the freezing-thawing procedure. 3 out of 4 proteins from isolated flagella were involved in the sperm bioenergetic system. Our results indicate that the ability of AFPIII to protect sea bream sperm quality can be, at least in part, ascribed to reducing changes in the sperm protein profile occurring during the freezing-thawing procedure. Our results clearly demonstrated that AFPIII addition to cryopreservation medium improved the protection against freezing respect to DMSO or DMSO plus AFPI. In addition we propose specific proteins of spermatozoa as markers related to

  10. Structure of solvation water around the active and inactive regions of a type III antifreeze protein and its mutants of lowered activity

    NASA Astrophysics Data System (ADS)

    Grabowska, Joanna; Kuffel, Anna; Zielkiewicz, Jan

    2016-08-01

    Water molecules from the solvation shell of the ice-binding surface are considered important for the antifreeze proteins to perform their function properly. Herein, we discuss the problem whether the extent of changes of the mean properties of solvation water can be connected with the antifreeze activity of the protein. To this aim, the structure of solvation water of a type III antifreeze protein from Macrozoarces americanus (eel pout) is investigated. A wild type of the protein is used, along with its three mutants, with antifreeze activities equal to 54% or 10% of the activity of the native form. The solvation water of the ice-binding surface and the rest of the protein are analyzed separately. To characterize the structure of solvation shell, parameters describing radial and angular characteristics of the mutual arrangement of the molecules were employed. They take into account short-distance (first hydration shell) or long-distance (two solvation shells) effects. The obtained results and the comparison with the results obtained previously for a hyperactive antifreeze protein from Choristoneura fumiferana lead to the conclusion that the structure and amino acid composition of the active region of the protein evolved to achieve two goals. The first one is the modification of the properties of the solvation water. The second one is the geometrical adjustment of the protein surface to the specific crystallographic plane of ice. Both of these goals have to be achieved simultaneously in order for the protein to perform its function properly. However, they seem to be independent from one another in a sense that very small antifreeze activity does not imply that properties of water become different from the ones observed for the wild type. The proteins with significantly lower activity still modify the mean properties of solvation water in a right direction, in spite of the fact that the accuracy of the geometrical match with the ice lattice is lost because of the

  11. Experimental investigation of the interactions of hyperactive antifreeze proteins with ice crystals

    NASA Astrophysics Data System (ADS)

    Celik, Yeliz

    Antifreeze proteins (AFPs) evolved in cold-adapted organisms and serve to protect them against freezing cold conditions by arresting ice crystal growth and inhibiting ice recrystallization. The freezing point depression by AFPs is defined as thermal hysteresis (TH) and AFPs are classified as hyperactive (hypAFPs) and moderate according to their TH activities. The mechanism of action of AFPs is not well understood. In particular, it is not clear what determines the concentration dependence of TH and whether the binding of AFP to ice is irreversible. Additionally, it is not known why some types of AFP are hyperactive compared to others and it was suggested that hyperactivity might be related to basal plane affinity of hypAFP to ice. The present study utilizes the techniques of microfluidic devices and fluorescence microscopy to study the interaction of AFPs with ice crystals. With novel temperature controlled microfluidic devices, we showed the accumulation and affinity of hypAFPs on the basal plane of ice. This supports the view that hypAFPs adhere to the basal plane. Additionally, for the first time in literature, small ice crystals of 30-50 mum sizes covered with adsorbed GFP tagged hypAFPs were stabilized in supercooled non-AFP solutions for hours with no observed ice growth in temperature controlled microfluidic devices. Repeated TH experiments of ice crystals incubated in AFP solutions before and after the exchange of liquids in microfluidic devices gave the same TH activity. This finding clarifies our understanding of concentration dependence of TH. Furthermore, we found that hypAFPs protect ice against melting as well as freezing, resulting in superheated ice. Ice crystals were superheated up to 0.5°C above their equilibrium melting temperatures and remained stable in this superheated state for hours. Measurements of fast melting velocities added additional evidence to the observed superheating of ice in AFP solutions. The experimental results of the current

  12. Molecular and comparative analyses of type IV antifreeze proteins (AFPIVs) from two Antarctic fishes, Pleuragramma antarcticum and Notothenia coriiceps.

    PubMed

    Lee, Jong Kyu; Kim, Yeon Ju; Park, Kyoung Sun; Shin, Seung Chul; Kim, Hak Jun; Song, Young Hwan; Park, Hyun

    2011-08-01

    Antifreeze protein type IV (AFPIV) cDNAs and genomic DNAs from the Antarctic fishes Pleuragramma antarcticum (Pa) and Notothenia coriiceps (Nc) were cloned and sequenced, respectively. Each cDNA encoded 128 amino acids, with 94% similarity between the two and 83% similarity with AFPIV of the longhorn sculpin, Myoxocephalus octodecemspinosus. The genome structures of both genes consisted of four exons and three introns, and were highly conserved in terms of sequences and positions. In contrast, the third intron of PaAFPIV had additional nucleotides with inverted repeats at each end, which appeared to be a MITE-like transposable element. Comparative analysis revealed that fish AFPIVs were widely distributed across teleost fishes, well conserved in their intron positions, but more variable in intron sequences and sizes. However, the intron sequences of two Antarctic fishes were highly conserved, indicating recent radiation of notothenioids in the evolutionary lineage. The recombinant PaAFPIV and NcAFPIV were expressed in E. coli, and examined antifreeze activity. PaAFPIV and NcAFPIV gave ice crystals with star-shaped morphology, and thermal hysteresis (TH) values were 0.08°C at the concentration of 0.5mg/ml.

  13. Gold Nanoparticle Aggregation as a Probe of Antifreeze (Glyco) Protein-Inspired Ice Recrystallization Inhibition and Identification of New IRI Active Macromolecules

    PubMed Central

    Mitchell, Daniel E.; Congdon, Thomas; Rodger, Alison; Gibson, Matthew I.

    2015-01-01

    Antifreeze (glyco)proteins are found in polar fish species and act to slow the rate of growth of ice crystals; a property known as ice recrystallization inhibition. The ability to slow ice growth is of huge technological importance especially in the cryopreservation of donor cells and tissue, but native antifreeze proteins are often not suitable, nor easily available. Therefore, the search for new materials that mimic this function is important, but currently limited by the low-throughout assays associated with the antifreeze properties. Here 30 nm gold nanoparticles are demonstrated to be useful colorimetric probes for ice recrystallization inhibition, giving a visible optical response and is compatible with 96 well plates for high-throughout studies. This method is faster, requires less infrastructure, and has easier interpretation than the currently used ‘splat’ methods. Using this method, a series of serum proteins were identified to have weak, but specific ice recrystallization inhibition activity, which was removed upon denaturation. It is hoped that high-throughput tools such as this will accelerate the discovery of new antifreeze mimics. PMID:26499135

  14. Gold Nanoparticle Aggregation as a Probe of Antifreeze (Glyco) Protein-Inspired Ice Recrystallization Inhibition and Identification of New IRI Active Macromolecules.

    PubMed

    Mitchell, Daniel E; Congdon, Thomas; Rodger, Alison; Gibson, Matthew I

    2015-10-26

    Antifreeze (glyco)proteins are found in polar fish species and act to slow the rate of growth of ice crystals; a property known as ice recrystallization inhibition. The ability to slow ice growth is of huge technological importance especially in the cryopreservation of donor cells and tissue, but native antifreeze proteins are often not suitable, nor easily available. Therefore, the search for new materials that mimic this function is important, but currently limited by the low-throughout assays associated with the antifreeze properties. Here 30 nm gold nanoparticles are demonstrated to be useful colorimetric probes for ice recrystallization inhibition, giving a visible optical response and is compatible with 96 well plates for high-throughout studies. This method is faster, requires less infrastructure, and has easier interpretation than the currently used 'splat' methods. Using this method, a series of serum proteins were identified to have weak, but specific ice recrystallization inhibition activity, which was removed upon denaturation. It is hoped that high-throughput tools such as this will accelerate the discovery of new antifreeze mimics.

  15. Expression of insect (Microdera puntipennis dzungarica) antifreeze protein MpAFP149 confers the cold tolerance to transgenic tobacco.

    PubMed

    Wang, Yan; Qiu, Liming; Dai, Chunying; Wang, Jing; Luo, Jianmin; Zhang, Fuchun; Ma, Ji

    2008-08-01

    To elucidate the function of antifreeze protein from Microdera puntipennis dzhungarica for freezing stress tolerance in plant, the construct of MpAFP149 gene with the signal peptide sequence responsible for secreting the native MpAFP149 into the apoplast space under control of a cauliflower mosaic virus 35S promoter was introduced into tobacco by Agrobacterium tumefaciens-mediated transformation. The observation of immunogold localization by TEM (transmission electron microscope) showed that the heterologous MpAFP149 protein was mainly distributed on the cell wall in apoplast of the transgenic tobacco plant. T1 generation transgenic tobacco plants displayed a more frost resistant phenotype and kept the lower ion leakage ratio and MDA (malondialdehyde) content in the leaves compared with wild-type ones at -1 degrees C for 3 days. The results showed that MpAFP149 provided protection and conferred cold tolerance to transgenic tobacco plant during freezing stress.

  16. Will It Be Beneficial To Simulate the Antifreeze Proteins at Ice Freezing Condition or at Lower Temperature?

    PubMed

    Kar, Rajiv K; Bhunia, Anirban

    2015-09-03

    Antifreeze proteins (AFPs) enable the polar living species to survive subzero temperature conditions through effective lowering of the freezing point of body fluids. At the molecular level, AFPs directly interact with the growing seeds of ice crystals to inhibit their formation. To understand the structural and dynamic aspects of this interaction at the atomistic level, molecular dynamics (MD) simulations were carried out on several type I AFPs at multiple temperatures, including the physiologically relevant temperature of 273 K, a lower temperature of 227 K, and the conventional 300 K. A comparison of the principal component analysis (PCA) and mean squared deviation plots for Winter flounder AFP, HPLC6 (mutant of winter flounder AFP), Sculpin, and peptide 1m AFPs reveals that simulations at 273 and 227 K result in the formation of more conserved metastable states than at 300 K. Other parameters such as root-mean-square deviation (rmsd), solvent accessibility surface area (SASA), H-bonding and residual density function (RDF) also suggest the same. MD simulations with ice crystal, where AFPs are complexed to ice plane with TIP4P/ice water model, help in finding relevance of dynamic behavior, and physiological temperature becomes more pronounced. Additionally, a control study on a nonantifreeze protein (LL37) is included, which aids in exploring significant information. On the basis of this approach, it was found that AFPs at 273 and 227 K display relevant dynamic properties that appear at 300 K for nonantifreeze proteins. The present study hence emphasizes the importance of performing computational simulations for antifreeze proteins at the physiologically relevant temperature (273 K), and even at lower temperatures (like 227 K), rather than at room temperatures (300 K).

  17. Effects of Three Different Types of Antifreeze Proteins on Mouse Ovarian Tissue Cryopreservation and Transplantation

    PubMed Central

    Youm, Hye Won; Kim, Hak Jun; Lee, Jung Ryeol; Suh, Chang Suk; Kim, Seok Hyun

    2015-01-01

    Background Ovarian tissue (OT) cryopreservation is effective in preserving fertility in cancer patients who have concerns about fertility loss due to cancer treatment. However, the damage incurred at different steps during the cryopreservation procedure may cause follicular depletion; hence, preventing chilling injury would help maintain ovarian function. Objective This study was designed to investigate the beneficial effects of different antifreeze proteins (AFPs) on mouse ovarian tissue cryopreservation and transplantation. Methodology Ovaries were obtained from 5-week-old B6D2F1 mice, and each ovary was cryopreserved using two-step vitrification and four-step warming procedures. In Experiment I, ovaries were randomly allocated into fresh, vitrification control, and nine experimental groups according to the AFP type (FfIBP, LeIBP, type III) and concentration (0.1, 1, 10 mg/mL) used. After vitrification and warming, 5,790 ovarian follicles were evaluated using histology and TUNEL assays, and immunofluorescence for τH2AX and Rad51 was used to detect DNA double-strand breaks (DSBs) and repair (DDR), respectively. In Experiment II, 20 mice were randomly divided into two groups: one where the vitrification and warming media were supplemented with 10 mg/mL LeIBP, and the other where media alone were used (control). Ovaries were then autotransplanted under both kidney capsules 7 days after vitrification together with the addition of 10 mg/mL LeIBP in the vitrification-warming media. After transplantation, the ovarian follicles, the percentage of apoptotic follicles, the extent of the CD31-positive area, and the serum FSH levels of the transplanted groups were compared. Principal Findings In Experiment I, the percentage of total grade 1 follicles was significantly higher in the 10 mg/mL LeIBP group than in the vitrification control, while all AFP-treated groups had significantly improved grade 1 primordial follicle numbers compared with those of the vitrification

  18. Transcriptomic and proteomic analyses on the supercooling ability and mining of antifreeze proteins of the Chinese white wax scale insect.

    PubMed

    Yu, Shu-Hui; Yang, Pu; Sun, Tao; Qi, Qian; Wang, Xue-Qing; Chen, Xiao-Ming; Feng, Ying; Liu, Bo-Wen

    2016-06-01

    The Chinese white wax scale insect, Ericerus pela, can survive at extremely low temperatures, and some overwintering individuals exhibit supercooling at temperatures below -30°C. To investigate the deep supercooling ability of E. pela, transcriptomic and proteomic analyses were performed to delineate the major gene and protein families responsible for the deep supercooling ability of overwintering females. Gene Ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that genes involved in the mitogen-activated protein kinase, calcium, and PI3K-Akt signaling pathways and pathways associated with the biosynthesis of soluble sugars, sugar alcohols and free amino acids were dominant. Proteins responsible for low-temperature stress, such as cold acclimation proteins, glycerol biosynthesis-related enzymes and heat shock proteins (HSPs) were identified. However, no antifreeze proteins (AFPs) were identified through sequence similarity search methods. A random forest approach identified 388 putative AFPs in the proteome. The AFP gene ep-afp was expressed in Escherichia coli, and the expressed protein exhibited a thermal hysteresis activity of 0.97°C, suggesting its potential role in the deep supercooling ability of E. pela.

  19. Cold survival in freeze-intolerant insects: the structure and function of beta-helical antifreeze proteins.

    PubMed

    Graether, Steffen P; Sykes, Brian D

    2004-08-01

    Antifreeze proteins (AFPs) designate a class of proteins that are able to bind to and inhibit the growth of macromolecular ice. These proteins have been characterized from a variety of organisms. Recently, the structures of AFPs from the spruce budworm (Choristoneura fumiferana) and the yellow mealworm (Tenebrio molitor) have been determined by NMR and X-ray crystallography. Despite nonhomologous sequences, both proteins were shown to consist of beta-helices. We review the structures and dynamics data of these two insect AFPs to bring insight into the structure-function relationship and explore their beta-helical architecture. For the spruce budworm protein, the fold is a left-handed beta-helix with 15 residues per coil. The Tenebrio molitor protein consists of a right-handed beta-helix with 12 residues per coil. Mutagenesis and structural studies show that the insect AFPs present a highly rigid array of threonine residues and bound water molecules that can effectively mimic the ice lattice. Comparisons of the newly determined ryegrass and carrot AFP sequences have led to models suggesting that they might also consist of beta-helices, and indicate that the beta-helix might be used as an AFP structural motif in nonfish organisms.

  20. FAST TRACK COMMUNICATION: Growth melt asymmetry in ice crystals under the influence of spruce budworm antifreeze protein

    NASA Astrophysics Data System (ADS)

    Pertaya, Natalya; Celik, Yeliz; Di Prinzio, Carlos L.; Wettlaufer, J. S.; Davies, Peter L.; Braslavsky, Ido

    2007-10-01

    Here we describe studies of the crystallization behavior of ice in an aqueous solution of spruce budworm antifreeze protein (sbwAFP) at atmospheric pressure. SbwAFP is an ice binding protein with high thermal hysteresis activity, which helps protect Choristoneura fumiferana (spruce budworm) larvae from freezing as they overwinter in the spruce and fir forests of the north eastern United States and Canada. Different types of ice binding proteins have been found in many other species. They have a wide range of applications in cryomedicine and cryopreservation, as well as the potential to protect plants and vegetables from frost damage through genetic engineering. However, there is much to learn regarding the mechanism of action of ice binding proteins. In our experiments, a solution containing sbwAFP was rapidly frozen and then melted back, thereby allowing us to produce small single crystals. These maintained their hexagonal shapes during cooling within the thermal hysteresis gap. Melt-growth-melt sequences in low concentrations of sbwAFP reveal the same shape transitions as are found in pure ice crystals at low temperature (-22 °C) and high pressure (2000 bar) (Cahoon et al 2006 Phys. Rev. Lett. 96 255502) while both growth and melt shapes display faceted hexagonal morphology, they are rotated 30° relative to one another. Moreover, the initial melt shape and orientation is recovered in the sequence. To visualize the binding of sbwAFP to ice, we labeled the antifreeze protein with enhanced green fluorescent protein (eGFP) and observed the sbwAFP-GFP molecules directly on ice crystals using confocal microscopy. When cooling the ice crystals, facets form on the six primary prism planes (slowest growing planes) that are evenly decorated with sbwAFP-GFP. During melting, apparent facets form on secondary prism planes (fastest melting planes), leaving residual sbwAFP at the six corners of the hexagon. Thus, the same general growth-melt behavior of an apparently rotated

  1. Distinct molecular features facilitating ice-binding mechanisms in hyperactive antifreeze proteins closely related to an Antarctic sea ice bacterium.

    PubMed

    Banerjee, Rachana; Chakraborti, Pratim; Bhowmick, Rupa; Mukhopadhyay, Subhasish

    2015-01-01

    Antifreeze proteins or ice-binding proteins (IBPs) facilitate the survival of certain cellular organisms in freezing environment by inhibiting the growth of ice crystals in solution. Present study identifies orthologs of the IBP of Colwellia sp. SLW05, which were obtained from a wide range of taxa. Phylogenetic analysis on the basis of conserved regions (predicted as the 'ice-binding domain' [IBD]) present in all the orthologs, separates the bacterial and archaeal orthologs from that of the eukaryotes'. Correspondence analysis pointed out that the bacterial and archaeal IBDs have relatively higher average hydrophobicity than the eukaryotic members. IBDs belonging to bacterial as well as archaeal AFPs contain comparatively more strands, and therefore are revealed to be under higher evolutionary selection pressure. Molecular docking studies prove that the ice crystals form more stable complex with the bacterial as well as archaeal proteins than the eukaryotic orthologs. Analysis of the docked structures have traced out the ice-binding sites (IBSs) in all the orthologs which continue to facilitate ice-binding activity even after getting mutated with respect to the well-studied IBSs of Typhula ishikariensis and notably, all these mutations performing ice-binding using 'anchored clathrate mechanism' have been found to prefer polar and hydrophilic amino acids. Horizontal gene transfer studies point toward a strong selection pressure favoring independent evolution of the IBPs in some polar organisms including prokaryotes as well as eukaryotes because these proteins facilitate the polar organisms to acclimatize to the adversities in their niche, thus safeguarding their existence.

  2. Effect of a mutation on the structure and dynamics of an alpha-helical antifreeze protein in water and ice.

    PubMed

    Graether, Steffen P; Slupsky, Carolyn M; Sykes, Brian D

    2006-05-15

    One strategy of psychrophilic organisms to survive subzero temperature is to produce antifreeze protein (AFPs), which inhibit the growth of macromolecular ice. To better understand the binding mechanism, the structure and dynamics of several AFPs have been studied by nuclear magnetic resonance (NMR) and X-ray crystallography. The results have shown that different organisms can use diverse structures (alpha-helix, beta-helix, or different globular folds) to achieve the same function. A number of studies have focused on understanding the relationship between the alpha-helical structure of fish type I AFP and its function as an inhibitor of ice growth. The results have not explained whether the 90% activity loss caused by the conservative mutation of two threonines to serines (Thr13Ser/Thr24Ser) is attributable to a change in protein structure in solution or in ice. We examine here the structure and dynamics of the winter flounder type I AFP and the mutant Thr13Ser/Thr24Ser in both solution and solid states using a wide range of NMR approaches. Both proteins remain fully alpha-helical at all temperatures and in ice, demonstrating that the activity change must therefore not be attributable to changes in the protein fold or dynamics but differences in surface properties.

  3. Antifreeze protein from freeze-tolerant grass has a beta-roll fold with an irregularly structured ice-binding site.

    PubMed

    Middleton, Adam J; Marshall, Christopher B; Faucher, Frédérick; Bar-Dolev, Maya; Braslavsky, Ido; Campbell, Robert L; Walker, Virginia K; Davies, Peter L

    2012-03-09

    The grass Lolium perenne produces an ice-binding protein (LpIBP) that helps this perennial tolerate freezing by inhibiting the recrystallization of ice. Ice-binding proteins (IBPs) are also produced by freeze-avoiding organisms to halt the growth of ice and are better known as antifreeze proteins (AFPs). To examine the structural basis for the different roles of these two IBP types, we have solved the first crystal structure of a plant IBP. The 118-residue LpIBP folds as a novel left-handed beta-roll with eight 14- or 15-residue coils and is stabilized by a small hydrophobic core and two internal Asn ladders. The ice-binding site (IBS) is formed by a flat beta-sheet on one surface of the beta-roll. We show that LpIBP binds to both the basal and primary-prism planes of ice, which is the hallmark of hyperactive AFPs. However, the antifreeze activity of LpIBP is less than 10% of that measured for those hyperactive AFPs with convergently evolved beta-solenoid structures. Whereas these hyperactive AFPs have two rows of aligned Thr residues on their IBS, the equivalent arrays in LpIBP are populated by a mixture of Thr, Ser and Val with several side-chain conformations. Substitution of Ser or Val for Thr on the IBS of a hyperactive AFP reduced its antifreeze activity. LpIBP may have evolved an IBS that has low antifreeze activity to avoid damage from rapid ice growth that occurs when temperatures exceed the capacity of AFPs to block ice growth while retaining the ability to inhibit ice recrystallization.

  4. Potential of mean force calculation of the free energy of adsorption of Type I winter flounder antifreeze protein on ice

    NASA Astrophysics Data System (ADS)

    Battle, Keith; Alan Salter, E.; Wesley Edmunds, R.; Wierzbicki, Andrzej

    2010-04-01

    Antifreeze proteins (AFPs) are a unique class of proteins that inhibit ice growth without changing the melting point of ice. In this work, we study the detailed molecular mechanism of interactions between the hydrophobic side of the winter flounder (WF) AFP and two mutants, AAAA and SSSS, in which threonine residues are substituted by serines and alanines, respectively. Umbrella sampling molecular dynamics simulations of the separation of the proteins from the (2 0 1) surface in an explicit water box is carried out to calculate the potential of mean force free energies of adsorption using AMBER10i. We estimate wild-type WF's free energy of adsorption to ice to be about -12.0 kcal/mol. Gas-phase pseudopotential plane-wave calculations of methane adsorption onto select surfaces of ice are also carried out under periodic boundary conditions to address the possible enthalpic role of WF's methyl groups in binding. The contributions of hydrophobic residues to the free energy of adsorption are discussed.

  5. Characterization of an antifreeze protein from the polar diatom Fragilariopsis cylindrus and its relevance in sea ice.

    PubMed

    Bayer-Giraldi, Maddalena; Weikusat, Ilka; Besir, Hüseyin; Dieckmann, Gerhard

    2011-12-01

    Antifreeze proteins (AFPs), characterized by their ability to separate the melting and growth temperatures of ice and to inhibit ice recrystallization, play an important role in cold adaptation of several polar and cold-tolerant organisms. Recently, a multigene family of AFP genes was found in the diatom Fragilariopsis cylindrus, a dominant species within polar sea ice assemblages. This study presents the AFP from F. cylindrus set in a molecular and crystallographic frame. Differential protein expression after exposure of the diatoms to environmentally relevant conditions underlined the importance of certain AFP isoforms in response to cold. Analyses of the recombinant AFP showed freezing point depression comparable to the activity of other moderate AFPs and further enhanced by salt (up to 0.9°C in low salinity buffer, 2.5°C at high salinity). However, unlike other moderate AFPs, its fastest growth direction is perpendicular to the c-axis. The protein also caused strong inhibition of recrystallization at concentrations of 1.2 and 0.12 μM at low and high salinity, respectively. Observations of crystal habit modifications and pitting activity suggested binding of AFPs to multiple faces of the ice crystals. Further analyses showed striations caused by AFPs, interpreted as inclusion in the ice. We suggest that the influence on ice microstructure is the main characteristic of these AFPs in sea ice.

  6. Molecular Characterization and Biological Effects of a C-Type Lectin-Like Receptor in Large Yellow Croaker (Larimichthys crocea)

    PubMed Central

    Ao, Jingqun; Ding, Yang; Chen, Yuanyuan; Mu, Yinnan; Chen, Xinhua

    2015-01-01

    The C-type lectin-like receptors (CTLRs) play important roles in innate immunity as one type of pattern recognition receptors. Here, we cloned and characterized a C-type lectin-like receptor (LycCTLR) from large yellow croaker Larimichthys crocea. The full-length cDNA of LycCTLR is 880 nucleotides long, encoding a protein of 215 amino acids. The deduced LycCTLR contains a C-terminal C-type lectin-like domain (CTLD), an N-terminal cytoplasmic tail, and a transmembrane region. The CTLD of LycCTLR possesses six highly conserved cysteine residues (C1–C6), a conserved WI/MGL motif, and two sugar binding motifs, EPD (Glu-Pro-Asp) and WYD (Trp-Tyr-Asp). Ca2+ binding site 1 and 2 were also found in the CTLD. The LycCTLR gene consists of five exons and four introns, showing the same genomic organization as tilapia (Oreochromis niloticus) and guppy (Poecilia retitculata) CTLRs. LycCTLR was constitutively expressed in various tissues tested, and its transcripts significantly increased in the head kidney and spleen after stimulation with inactivated trivalent bacterial vaccine. Recombinant LycCTLR (rLycCTLR) protein produced in Escherichia coli BL21 exhibited not only the hemagglutinating activity and a preference for galactose, but also the agglutinating activity against two food-borne pathogenic bacteria E. coli and Bacillus cereus in a Ca2+-dependent manner. These results indicate that LycCTLR is a potential galactose-binding C-type lectin that may play a role in the antibacterial immunity in fish. PMID:26690423

  7. Ice-binding site of snow mold fungus antifreeze protein deviates from structural regularity and high conservation.

    PubMed

    Kondo, Hidemasa; Hanada, Yuichi; Sugimoto, Hiroshi; Hoshino, Tamotsu; Garnham, Christopher P; Davies, Peter L; Tsuda, Sakae

    2012-06-12

    Antifreeze proteins (AFPs) are found in organisms ranging from fish to bacteria, where they serve different functions to facilitate survival of their host. AFPs that protect freeze-intolerant fish and insects from internal ice growth bind to ice using a regular array of well-conserved residues/motifs. Less is known about the role of AFPs in freeze-tolerant species, which might be to beneficially alter the structure of ice in or around the host. Here we report the 0.95-Å high-resolution crystal structure of a 223-residue secreted AFP from the snow mold fungus Typhula ishikariensis. Its main structural element is an irregular β-helix with six loops of 18 or more residues that lies alongside an α-helix. β-Helices have independently evolved as AFPs on several occasions and seem ideally structured to bind to several planes of ice, including the basal plane. A novelty of the β-helical fold is the nonsequential arrangement of loops that places the N- and C termini inside the solenoid of β-helical coils. The ice-binding site (IBS), which could not be predicted from sequence or structure, was located by site-directed mutagenesis to the flattest surface of the protein. It is remarkable for its lack of regularity and its poor conservation in homologs from psychrophilic diatoms and bacteria and other fungi.

  8. Purification of antifreeze protein from wheat bran (Triticum aestivum L.) based on its hydrophilicity and ice-binding capacity.

    PubMed

    Zhang, Chao; Zhang, Hui; Wang, Li; Zhang, Junhui; Yao, Huiyuan

    2007-09-19

    Wheat-bran ( Triticum aestivum L.) antifreeze protein ( TaAFP) was purified 323-fold to electrophoretic homogeneity with an overall yield of 1.64% from wheat-bran protein by a specific three-step procedure. The three-step procedure was quicker, cheaper, and more effective than the five-step procedure we used earlier. First, TaAFP was concentrated by a phosphate buffer, on the basis of its strong hydrophilicity that was validated by thermal gravimetric analyses and a surface hydrophobicity analysis. Second, TaAFP was trapped in ice crystals for its specific ice-binding capacity, which was proved by ice-binding protocols. Remarkably, the ice-binding step was the most effective step, and the purification factor of this step was up to 270-fold. Finally, TaAFP was purified by HPLC purification, a complementary step for the specific ice-binding protocol, to electrophoretic homogeneity. Our protocols provide peers a novel and effective way for the search and purification of potential AFPs.

  9. Purification, crystal structure determination and functional characterization of type III antifreeze proteins from the European eelpout Zoarces viviparus.

    PubMed

    Wilkens, Casper; Poulsen, Jens-Christian N; Ramløv, Hans; Lo Leggio, Leila

    2014-08-01

    Antifreeze proteins (AFPs) are essential components of many organisms adaptation to cold temperatures. Fish type III AFPs are divided into two groups, SP isoforms being much less active than QAE1 isoforms. Two type III AFPs from Zoarces viviparus, a QAE1 (ZvAFP13) and an SP (ZvAFP6) isoform, are here characterized and their crystal structures determined. We conclude that the higher activity of the QAE1 isoforms cannot be attributed to single residues, but rather a combination of structural effects. Furthermore both ZvAFP6 and ZvAFP13 crystal structures have water molecules around T18 equivalent to the tetrahedral-like waters previously identified in a neutron crystal structure. Interestingly, ZvAFP6 forms dimers in the crystal, with a significant dimer interface. The presence of ZvAFP6 dimers was confirmed in solution by native electrophoresis and gel filtration. To our knowledge this is the first report of dimerization of AFP type III proteins.

  10. O serotype-independent susceptibility of Pseudomonas aeruginosa to lectin-like pyocins

    PubMed Central

    Ghequire, Maarten G K; Dingemans, Jozef; Pirnay, Jean-Paul; De Vos, Daniel; Cornelis, Pierre; De Mot, René

    2014-01-01

    Lectin-like bacteriocins of the LlpA family, originally identified in plant-associated bacteria, are narrow-spectrum antibacterial proteins composed of two tandemly organized monocot mannose-binding lectin (MMBL) domains. The LlpA-like bacteriocin of Pseudomonas aeruginosa C1433, pyocin L1, lacks any similarity to known P. aeruginosa bacteriocins. The initial interaction of pyocin L1 with target cells is mediated by binding to d-rhamnose, present in the common polysaccharide antigen of lipopolysaccharides (LPS), but the actual cytotoxic mechanism is unknown. In this study, we characterized the activity range of pyocin L1 and two additional L pyocins revealed by genome mining, representing two highly diverged LlpA groups in P. aeruginosa. The recombinant proteins exhibit species-specific antagonistic activities down to nanomolar concentrations against clinical and environmental P. aeruginosa strains, including several multidrug-resistant isolates. The overlap in target strain spectrum between two close homologues of the pyocin L1 group is only minimal, contrasting with the considerable spectral redundancy of LlpA proteins reported for other Pseudomonas species. No correlation was found between L pyocin susceptibility and phylogenetic relatedness of P. aeruginosa isolates. Sensitive strains were retrieved in 13 out of 15 O serotypes tested, excluding the possibility that the highly variable and immunogenic O serotype antigen of the LPS coating would represent a dominant susceptibility-discriminating factor. PMID:25224846

  11. Structure and Dynamics of Antifreeze Protein--Model Membrane Interactions: A Combined Spectroscopic and Molecular Dynamics Study.

    PubMed

    Kar, Rajiv K; Mroue, Kamal H; Kumar, Dinesh; Tejo, Bimo A; Bhunia, Anirban

    2016-02-11

    Antifreeze proteins (AFPs) are the key biomolecules that enable species to survive under subzero temperature conditions. The physiologically relevant activities of AFPs are based on the adsorption to ice crystals, followed by the inhibition of subsequent crystal layer growth of ice, routed with depression in freezing point in a noncolligative manner. The functional attributes governing the mechanism by which AFPs inhibit freezing of body fluids in bacteria, fungi, plants, and fishes are mainly attributed to their adsorption onto the surface of ice within the physiological system. Importantly, AFPs are also known for their application in cryopreservation of biological samples that might be related to membrane interaction. To date, there is a paucity of information detailing the interaction of AFPs with membrane structures. Here, we focus on elucidating the biophysical properties of the interactions between AFPs and micelle models that mimic the membrane system. Micelle model systems of zwitterionic DPC and negatively charged SDS were utilized in this study, against which a significant interaction is experienced by two AFP molecules, namely, Peptide 1m and wfAFP (the popular AFP sourced from winter flounder). Using low- and high-resolution biophysical characterization techniques, such as circular dichroism (CD) and NMR spectroscopy, a strong evidence for the interactions of these AFPs with the membrane models is revealed in detail and is corroborated by in-depth residue-specific information derived from molecular dynamics simulation. Altogether, these results not only strengthen the fact that AFPs interact actively with membrane systems, but also demonstrate that membrane-associated AFPs are dynamic and capable of adopting a number of conformations rendering fluidity to the system.

  12. Effect of the antifreeze protein from the arctic yeast Leucosporidium sp. AY30 on cryopreservation of the marine diatom Phaeodactylum tricornutum.

    PubMed

    Koh, Hye Yeon; Lee, Jun Hyuck; Han, Se Jong; Park, Hyun; Lee, Sung Gu

    2015-01-01

    Antifreeze proteins are a group of proteins that allow organisms to survive in subzero environments. These proteins possess thermal hysteresis and ice recrystallization inhibition activities. In the present study, we demonstrated the efficiency of a recombinant antifreeze protein from the Arctic yeast Leucosporidium sp. AY30, LeIBP, in cryopreservation of the marine diatom Phaeodactylum tricornutum, which is one of the classical model diatoms and has most widely been studied with regard to its ecology, physiology, biochemistry, and molecular biology. P. tricornutum cells were frozen by either a fast or two-step freezing method in freezing medium containing 10 % dimethyl sulfoxide, glycerol, propylene glycol, and ethylene glycol, respectively, with or without LeIBP supplement. When cells were frozen using the two-step freezing method, cell survival was significantly increased and statistically the same as that of unfrozen native cells in the presence of 0.1 mg/ml LeIBP in 10 % propylene glycol or 10 % ethylene glycol at day 11 of post-thaw culture. In the presence of LeIBP, the concentration of chlorophyll a was dramatically increased to 14-, 48-, 1.6-, and 8.8-fold when cells were frozen in freezing medium containing dimethyl sulfoxide (DMSO), glycerol, propylene glycol (PG), and ethylene glycol (EG), respectively. Scanning electron microscopy observations demonstrated that the cells were also successfully preserved and epitheca or hypotheca were not deformed. These results demonstrate that LeIBP was successfully applied to improve cryopreservation of the marine diatom P. tricornutum.

  13. Structure-function relationship in the antifreeze activity of synthetic alanine-lysine antifreeze polypeptides.

    PubMed

    Wierzbicki, A; Knight, C A; Rutland, T J; Muccio, D D; Pybus, B S; Sikes, C S

    2000-01-01

    Recently antifreeze proteins (AFP) have been the subject of many structure-function relationship studies regarding their antifreeze activity. Attempts have been made to elucidate the structure-function relationship by various amino acid substitutions, but to our knowledge there has been no successful from first principles design of a polypeptide that would bind to designated ice planes along a specific direction. In this paper we show the results of our first attempt on an entirely de novo design of an alanine-lysine-rich antifreeze polypeptide. This 43 residue alanine-lysine peptide exhibits characteristic nonequilibrium freezing point depression and binds to the designated (210) planes of ice along the [122] vector. The structural and thermodynamic properties of this polypeptide were determined using circular dichroism spectroscopy and its nonequilibrium antifreeze properties were investigated using an ice-etching method and nanoliter osmometry.

  14. Developmental and environmental regulation of antifreeze proteins in the mealworm beetle Tenebrio molitor.

    PubMed

    Graham, L A; Walker, V K; Davies, P L

    2000-11-01

    The yellow mealworm beetle, Tenebrio molitor, contains a family of small Cys-rich and Thr-rich thermal hysteresis proteins that depress the hemolymph freezing point below the melting point by as much as 5. 5 degrees C (DeltaT = thermal hysteresis). Thermal hysteresis protein expression was evaluated throughout development and after exposure to altered environmental conditions. Under favorable growth conditions, small larvae (11-13 mg) had only low levels of thermal hysteresis proteins or thermal hysteresis protein message, but these levels increased 10-fold and 18-fold, respectively, by the final larval instar (> 190 mg), resulting in thermal hysteresis > 3 degrees C. Exposure of small larvae (11-13 mg) to 4 weeks of cold (4 degrees C) caused an approximately 20-fold increase in thermal hysteresis protein concentration, well in excess of the less than threefold developmental increase seen after 4 weeks at 22 degrees C. Exposure of large larvae (100-120 mg) to cold caused 12-fold and sixfold increases in thermal hysteresis protein message and protein levels, respectively, approximately double the maximum levels they would have attained in the final larval instar at 22 degrees C. Thus, thermal hysteresis increased to similar levels (> 4 degrees C) in the cold, irrespective of the size of the larvae (the overwintering stage). At pupation, thermal hysteresis protein message levels decreased > 20-fold and remained low thereafter, but thermal hysteresis activity decreased much more slowly. Exposure to cold did not reverse this decline. Desiccation or starvation of larvae had comparable effects to cold exposure, but surprisingly, short daylength photoperiod or total darkness had no effect on either thermal hysteresis or message levels. As all environmental conditions that caused increased thermal hysteresis also inhibited growth, we postulate that developmental arrest is a primary factor in the regulation of T. molitor thermal hysteresis proteins.

  15. Saccharide antifreeze compositions

    DOEpatents

    Walters, Kent; Duman, John G; Serianni, Anthony S

    2013-12-10

    The invention provides an antifreeze glycolipid compounds and composition comprising a polysaccharide moiety of Formula I; ##STR00001## wherein D-Manp represents a D-mannopyranose moiety, D-Xylp represents a D-xylopyranose moiety, and n is about 5 to about 70; and one or more lipid moieties covalently linked to the polysaccharide moiety of Formula I or electrostatically associated with the polysaccaride moiety for Formula I. The antifreeze glycolipid compounds and compositions can be used for a variety of industrial, agricultural, medical, and cosmetic applications where recrystallization-inhibition, cyroprotection, or cryopreservation is desired. The antifreeze glycolipid compounds or compositions can be used as, for example, as cryoprotectants for tissue preservation and transplantation, improving the texture of processed frozen food and frozen meats, frostbit protection, crop protection, and green alternatives for land vehicle antifreeze and aircraft de-icing.

  16. C-type lectin-like receptors of the dectin-1 cluster: ligands and signaling pathways.

    PubMed

    Plato, Anthony; Willment, Janet A; Brown, Gordon D

    2013-04-01

    Innate immunity is constructed around genetically encoded receptors that survey the intracellular and extracellular environments for signs of invading microorganisms. These receptors recognise the invader and through complex intracellular networks of molecular signaling, they destroy the threat whilst instructing effective adaptive immune responses. Many of these receptors, like the Toll-like receptors in particular, are well-known for their ability to mediate downstream responses upon recognition of exogenous or endogenous ligands; however, the emerging family known as the C-type lectin-like receptors contains many members that have a huge impact on immune and homeostatic regulation. Of particular interest here are the C-type lectin-like receptors that make up the Dectin-1 cluster and their intracellular signaling motifs that mediate their functions. In this review, we aim to draw together current knowledge of ligands, motifs and signaling pathways, present downstream of Dectin-1 cluster receptors, and discuss how these dictate their role within biological systems.

  17. Structure-based characterization and antifreeze properties of a hyperactive ice-binding protein from the Antarctic bacterium Flavobacterium frigoris PS1.

    PubMed

    Do, Hackwon; Kim, Soon-Jong; Kim, Hak Jun; Lee, Jun Hyuck

    2014-04-01

    Ice-binding proteins (IBPs) inhibit ice growth through direct interaction with ice crystals to permit the survival of polar organisms in extremely cold environments. FfIBP is an ice-binding protein encoded by the Antarctic bacterium Flavobacterium frigoris PS1. The X-ray crystal structure of FfIBP was determined to 2.1 Å resolution to gain insight into its ice-binding mechanism. The refined structure of FfIBP shows an intramolecular disulfide bond, and analytical ultracentrifugation and analytical size-exclusion chromatography show that it behaves as a monomer in solution. Sequence alignments and structural comparisons of IBPs allowed two groups of IBPs to be defined, depending on sequence differences between the α2 and α4 loop regions and the presence of the disulfide bond. Although FfIBP closely resembles Leucosporidium (recently re-classified as Glaciozyma) IBP (LeIBP) in its amino-acid sequence, the thermal hysteresis (TH) activity of FfIBP appears to be tenfold higher than that of LeIBP. A comparison of the FfIBP and LeIBP structures reveals that FfIBP has different ice-binding residues as well as a greater surface area in the ice-binding site. Notably, the ice-binding site of FfIBP is composed of a T-A/G-X-T/N motif, which is similar to the ice-binding residues of hyperactive antifreeze proteins. Thus, it is proposed that the difference in TH activity between FfIBP and LeIBP may arise from the amino-acid composition of the ice-binding site, which correlates with differences in affinity and surface complementarity to the ice crystal. In conclusion, this study provides a molecular basis for understanding the antifreeze mechanism of FfIBP and provides new insights into the reasons for the higher TH activity of FfIBP compared with LeIBP.

  18. Lysophosphatidic acid stimulates thrombomodulin lectin-like domain shedding in human endothelial cells

    SciTech Connect

    Wu Hualin; Lin ChiIou; Huang Yuanli; Chen, Pin-Shern; Kuo, Cheng-Hsiang; Chen, Mei-Shing; Wu, G.C.-C.; Shi, G.-Y.; Yang, H.-Y.; Lee Hsinyu

    2008-02-29

    Thrombomodulin (TM) is an anticoagulant glycoprotein highly expressed on endothelial cell surfaces. Increased levels of soluble TM in circulation have been widely accepted as an indicator of endothelial damage or dysfunction. Previous studies indicated that various proinflammatory factors stimulate TM shedding in various cell types such as smooth muscle cells and epithelial cells. Lysophosphatidic acid (LPA) is a bioactive lipid mediator present in biological fluids during endothelial damage or injury. In the present study, we first observed that LPA triggered TM shedding in human umbilical vein endothelial cells (HUVECs). By Cyflow analysis, we showed that the LPA-induced accessibility of antibodies to the endothelial growth factor (EGF)-like domain of TM is independent of matrix metalloproteinases (MMPs), while LPA-induced TM lectin-like domain shedding is MMP-dependent. Furthermore, a stable cell line expressing TM without its lectin-like domain exhibited a higher cell proliferation rate than a stable cell line expressing full-length TM. These results imply that LPA induces TM lectin-like domain shedding, which might contribute to the exposure of its EGF-like domain for EGF receptor (EGFR) binding, thereby stimulating subsequent cell proliferation. Based on our findings, we propose a novel mechanism for the exposure of TM EGF-like domain, which possibly mediates LPA-induced EGFR transactivation.

  19. Identification and Evaluation of Cryoprotective Peptides from Chicken Collagen: Ice-Growth Inhibition Activity Compared to That of Type I Antifreeze Proteins in Sucrose Model Systems.

    PubMed

    Du, Lihui; Betti, Mirko

    2016-06-29

    The ability of chicken collagen peptides to inhibit the growth of ice crystals was evaluated and compared to that of fish antifreeze proteins (AFPs). This ice inhibition activity was assessed using a polarized microscope by measuring ice crystal dimensions in a sucrose model system with and without collagen peptides after seven thermal cycles. The system was stabilized at -25 °C and cycled between -16 and -12 °C. Five candidate peptides with ice inhibition activity were identified using liquid chromatography and tandem mass spectrometry and were then synthesized. Their ice inhibition capacity was compared to that of type I AFPs in a 23% sucrose model system. Specific collagen peptides with certain amino acid sequences reduced the extent of ice growth by approximately 70% at a relatively low concentration (1 mg/mL). These results suggest that specific collagen peptides may act in a noncolligative manner, inhibiting ice crystal growth like type I AFPs, but less efficiently.

  20. Computational study on the function of water within a beta-helix antifreeze protein dimer and in the process of ice-protein binding.

    PubMed

    Yang, Zuoyin; Zhou, Yanxia; Liu, Kai; Cheng, Yuhua; Liu, Ruozhuang; Chen, Guangju; Jia, Zongchao

    2003-10-01

    Antifreeze proteins (AFPs) help many organisms protect themselves from freezing in subzero temperatures. The most active AFPs found to date are those from insects, which possess exceptionally regular beta-helical structures. On the ice-binding surface of these proteins, regularly arrayed water molecules are observed within the repeating Thr-Xxx-Thr motif, but the exact role of these water molecules remains unknown. In this work, we have employed a number of computational methods to examine the role of these water molecules in an AFP from Tenebrio molitor (TmAFP). Our investigation involved a combination of molecular and quantum mechanical approaches. Properties such as stability, interaction energy, orbital overlap, and conformational analysis of various systems, including TmAFP-water, TmAFP-water-ice, and TmAFP-ice, were systematically evaluated and compared. The regularly arrayed water molecules were found to remain associated with TmAFP before ice binding, demonstrating that they are an intrinsic part of the protein. These water molecules may assist TmAFP in the process of ice recognition and binding. However, after facilitating the initial stages of ice recognition and binding, these water molecules are excluded in the final formation of the AFP-ice complex. The departure of these water molecules enables a better two-dimensional match between TmAFP and ice. These results agree with experimental observations showing that although these water molecules are aligned with the ice-binding hydroxyl groups of Thr residues in one dimension, they are in fact positioned slightly off in the second dimension, making a good two-dimensional match impossible.

  1. Engulfment and clearance of apoptotic cells based on a GlcNAc-binding lectin-like property of surface vimentin.

    PubMed

    Ise, Hirohiko; Goto, Mitsuaki; Komura, Kenta; Akaike, Toshihiro

    2012-06-01

    The clearance of apoptotic cells is important to maintain tissue homeostasis. The engulfment of apoptotic cells is performed by professional phagocytes, such as macrophages, and also by non-professional phagocytes, such as mesenchymal cells. Here, we show that vimentin, a cytoskeletal protein, functions as an engulfment receptor on neighboring phagocytes, which recognize O-linked β-N-acetylglucosamine (O-GlcNAc)-modified proteins from apoptotic cells as "eat me" ligands. Previously, we reported that vimentin possesses a GlcNAc-binding lectin-like property on cell surface. However, the physiological relevance of the surface localization and GlcNAc-binding property of vimentin remained unclear. In the present study, we observed that O-GlcNAc proteins from apoptotic cells interacted with the surface vimentin of neighboring phagocytes and that this interaction induced serine 71-phosphorylation and recruitment of vimentin to the cell surface of the neighboring phagocytes. Moreover, tetrameric vimentin that was disassembled by serine 71-phosphorylation possessed a GlcNAc-binding activity and was localized to the cell surface. We demonstrated our findings in vimentin-expressing common cell lines such as HeLa cells. Furthermore, during normal developmental processes, the phagocytic engulfment and clearance of apoptotic footplate cells in mouse embryos was mediated by the interaction of surface vimentin with O-GlcNAc proteins. Our results suggest a common mechanism for the clearance of apoptotic cells, through the interaction of surface vimentin with O-GlcNAc-modified proteins.

  2. iAFP-Ense: An Ensemble Classifier for Identifying Antifreeze Protein by Incorporating Grey Model and PSSM into PseAAC.

    PubMed

    Xiao, Xuan; Hui, Mengjuan; Liu, Zi

    2016-12-01

    Antifreeze proteins (AFPs), known as thermal hysteresis proteins, are ice-binding proteins. AFPs have been found in many fields such as in vertebrates, invertebrates, plants, bacteria, and fungi. Although the function of AFPs is common, the sequences and structures of them show a high degree of diversity. AFPs can be adsorbed in ice crystal surface and inhibit the growth of ice crystals in solution. However, the interaction between AFPs and ice crystal is not completely known for human beings. It is vitally significant to propose an automated means as a high-throughput tool to timely identify the AFPs. Analyzing physicochemical characteristics of AFPs sequences is very significant to understand the ice-protein interaction. In this manuscript, a predictor called "iAFP-Ense" was developed. The operation engine to run the AFPs prediction is an ensemble classifier formed by a voting system to fuse eleven different random forest classifiers based on feature extraction. We also compare our predictor with the AFP-PseAAC via the tenfold cross-validation on the same benchmark dataset. The comparison with the existing methods indicates the new predictor is very promising, meaning that many important key features which are deeply hidden in complicated protein sequences. The predictor used in this article is freely available at http://www.jci-bioinfo.cn/iAFP-Ense .

  3. Rational, yet simple, design and synthesis of an antifreeze-protein inspired polymer for cellular cryopreservation† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c5cc04647e Click here for additional data file.

    PubMed Central

    Mitchell, Daniel E.; Cameron, Neil R.

    2015-01-01

    Antifreeze (glyco) proteins AF(G)Ps are potent ice recrystallization inhibitors, which is a desirable property to enhance cryopreservation of donor tissue/cells. Here we present the rational synthesis of a new, biomimetic, ice-recrystallization inhibiting polymer derived from a cheap commodity polymer, based on an ampholyte structure. The polymer is used to enhance the cryopreservation of red blood cells, demonstrating a macromolecular solution to tissue storage. PMID:26176027

  4. Antifreeze glycopeptide diastereomers.

    PubMed

    Nagel, Lilly; Budke, Carsten; Dreyer, Axel; Koop, Thomas; Sewald, Norbert

    2012-01-01

    Antifreeze glycopeptides (AFGPs) are a special class of biological antifreeze agents, which possess the property to inhibit ice growth in the body fluids of arctic and antarctic fish and, thus, enable life under these harsh conditions. AFGPs are composed of 4-55 tripeptide units -Ala-Ala-Thr- glycosylated at the threonine side chains. Despite the structural homology among all the fish species, divergence regarding the composition of the amino acids occurs in peptides from natural sources. Although AFGPs were discovered in the early 1960s, the adsorption mechanism of these macromolecules to the surface of the ice crystals has not yet been fully elucidated. Two AFGP diastereomers containing different amino acid configurations were synthesized to study the influence of amino acid stereochemistry on conformation and antifreeze activity. For this purpose, peptides containing monosaccharide-substituted allo-L- and D-threonine building blocks were assembled by solid-phase peptide synthesis (SPPS). The retro-inverso AFGP analogue contained all amino acids in D-configuration, while the allo-L-diastereomer was composed of L-amino acids, like native AFGPs, with replacement of L-threonine by its allo-L-diastereomer. Both glycopeptides were analyzed regarding their conformational properties, by circular dichroism (CD), and their ability to inhibit ice recrystallization in microphysical experiments.

  5. Antifreeze glycopeptide diastereomers

    PubMed Central

    Nagel, Lilly; Budke, Carsten; Dreyer, Axel; Koop, Thomas

    2012-01-01

    Summary Antifreeze glycopeptides (AFGPs) are a special class of biological antifreeze agents, which possess the property to inhibit ice growth in the body fluids of arctic and antarctic fish and, thus, enable life under these harsh conditions. AFGPs are composed of 4–55 tripeptide units -Ala-Ala-Thr- glycosylated at the threonine side chains. Despite the structural homology among all the fish species, divergence regarding the composition of the amino acids occurs in peptides from natural sources. Although AFGPs were discovered in the early 1960s, the adsorption mechanism of these macromolecules to the surface of the ice crystals has not yet been fully elucidated. Two AFGP diastereomers containing different amino acid configurations were synthesized to study the influence of amino acid stereochemistry on conformation and antifreeze activity. For this purpose, peptides containing monosaccharide-substituted allo-L- and D-threonine building blocks were assembled by solid-phase peptide synthesis (SPPS). The retro-inverso AFGP analogue contained all amino acids in D-configuration, while the allo-L-diastereomer was composed of L-amino acids, like native AFGPs, with replacement of L-threonine by its allo-L-diastereomer. Both glycopeptides were analyzed regarding their conformational properties, by circular dichroism (CD), and their ability to inhibit ice recrystallization in microphysical experiments. PMID:23209499

  6. Validation of antifreeze properties of glutathione based on its thermodynamic characteristics and protection of baker's yeast during cryopreservation.

    PubMed

    Zhang, Chao; Zhang, Hui; Wang, Li; Yao, Huiyuan

    2007-06-13

    The antifreeze ability of glutathione was evaluated on the basis of its thermodynamic characteristics and protection of baker's yeast during cryopreservation at -30 degrees C. The thermodynamic characteristics and protection of baker's yeast of glutathione were similar to those of known antifreeze proteins, such as carrot antifreeze protein and holly antifreeze protein. These properties included lowering the freezing point at about 0.20 degrees C non-colligatively, decreasing freezable water content, controlling the movement of free water for its strong hydrophilicity, and improving baker's yeast survival during the simulated processing of frozen dough. Therefore, glutathione was viewed to be an antifreeze protein like substance on the basis of its unique thermodynamic characteristics and protection of baker's yeast. The method combining thermodynamic characteristic analysis and protection evaluation is a new and simple way to screen new antifreeze proteins.

  7. Lectin-like oxidized low-density lipoprotein receptor (LOX-1) in sickle cell disease vasculopathy.

    PubMed

    Chen, Mingyi; Qiu, Hong; Lin, Xin; Nam, David; Ogbu-Nwobodo, Lucy; Archibald, Hannah; Joslin, Amelia; Wun, Ted; Sawamura, Tatsuya; Green, Ralph

    2016-09-01

    Lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (LOX-1) is an endothelial receptor for oxidized LDL. Increased expression of LOX-1 has been demonstrated in atherosclerotic lesions and diabetic vasculopathy. In this study, we investigate the expression of LOX-1 receptor in sickle cell disease (SCD) vasculopathy. Expression of LOX-1 in brain vascular endothelium is markedly increased and LOX-1 gene expression is upregulated in cultured human brain microvascular endothelial cells by incubation with SCD erythrocytes. Also, the level of circulating soluble LOX-1 concentration is elevated in the plasma of SCD patients. Increased LOX-1 expression in endothelial cells is potentially involved in the pathogenesis of SCD vasculopathy. Soluble LOX-1 concentration in SCD may provide a novel biomarker for risk stratification of sickle cell vascular complications.

  8. Lectin-like oxidized low-density lipoprotein receptor (LOX-1) in sickle cell disease vasculopathy

    PubMed Central

    Chen, Mingyi; Qiu, Hong; Lin, Xin; Nam, David; Ogbu-Nwobodo, Lucy; Archibald, Hannah; Joslin, Amelia; Wun, Ted; Sawamura, Tatsuya; Green, Ralph

    2017-01-01

    Lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (LOX-1) is an endothelial receptor for oxidized LDL. Increased expression of LOX-1 has been demonstrated in atherosclerotic lesions and diabetic vasculopathy. In this study, we investigate the expression of LOX-1 receptor in sickle cell disease (SCD) vasculopathy. Expression of LOX-1 in brain vascular endothelium is markedly increased and LOX-1 gene expression is upregulated in cultured human brain microvascular endothelial cells by incubation with SCD erythrocytes. Also, the level of circulating soluble LOX-1 concentration is elevated in the plasma of SCD patients. Increased LOX-1 expression in endothelial cells is potentially involved in the pathogenesis of SCD vasculopathy. Soluble LOX-1 concentration in SCD may provide a novel biomarker for risk stratification of sickle cell vascular complications. PMID:27519944

  9. C-type lectin-like receptor 2 promotes hematogenous tumor metastasis and prothrombotic state in tumor-bearing mice.

    PubMed

    Shirai, T; Inoue, O; Tamura, S; Tsukiji, N; Sasaki, T; Endo, H; Satoh, K; Osada, M; Sato-Uchida, H; Fujii, H; Ozaki, Y; Suzuki-Inoue, K

    2017-03-01

    Essentials The role of C-type lectin-like receptor-2 (CLEC-2) in cancer progression is unclear. CLEC-2-depleted mouse model is generated by using a rat anti-mouse CLEC-2 monoclonal antibody. CLEC-2 depletion inhibits hematogenous tumor metastasis of podoplanin-expressing B16F10 cells. CLEC-2 depletion prolongs cancer survival by suppressing thrombosis and inflammation.

  10. Antifreeze glycopeptide adsorption on single crystal ice surfaces using ellipsometry

    PubMed Central

    Wilson, P. W.; Beaglehole, D.; DeVries, A. L.

    1993-01-01

    Antarctic fishes synthesise antifreeze proteins which can effectively inhibit the growth of ice crystals. The mechanism relies on adsorption of these proteins to the ice surface. Ellipsometry has been used to quantify glycopeptide antifreeze adsorption to the basal and prism faces of single ice crystals. The rate of accumulation was determined as a function of time and at concentrations between 0.0005 and 1.2 mg/ml. Estimates of packing density at saturation coverage have been made for the basal and prism faces. PMID:19431902

  11. Seasonal freeze resistance of rainbow smelt (Osmerus mordax) is generated by differential expression of glycerol-3-phosphate dehydrogenase, phosphoenolpyruvate carboxykinase, and antifreeze protein genes.

    PubMed

    Liebscher, Ryan S; Richards, Robert C; Lewis, Johanne M; Short, Connie E; Muise, Denise M; Driedzic, William R; Ewart, K Vanya

    2006-01-01

    In winter, rainbow smelt (Osmerus mordax) accumulate glycerol and produce an antifreeze protein (AFP), which both contribute to freeze resistance. The role of differential gene expression in the seasonal pattern of these adaptations was investigated. First, cDNAs encoding smelt and Atlantic salmon (Salmo salar) phosphoenolpyruvate carboxykinase (PEPCK) and smelt glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were cloned so that all sequences required for expression analysis would be available. Using quantitative PCR, expression of beta actin in rainbow smelt liver was compared with that of GAPDH in order to determine its validity as a reference gene. Then, levels of glycerol-3-phosphate dehydrogenase (GPDH), PEPCK, and AFP relative to beta actin were measured in smelt liver over a fall-winter-spring interval. Levels of GPDH mRNA increased in the fall just before plasma glycerol accumulation, implying a driving role in glycerol synthesis. GPDH mRNA levels then declined during winter, well in advance of serum glycerol, suggesting the possibility of GPDH enzyme or glycerol conservation in smelt during the winter months. PEPCK mRNA levels rose in parallel with serum glycerol in the fall, consistent with an increasing requirement for amino acids as metabolic precursors, remained elevated for much of the winter, and then declined in advance of the decline in plasma glycerol. AFP mRNA was elevated at the onset of fall sampling in October and remained elevated until April, implying separate regulation from GPDH and PEPCK. Thus, winter freezing point depression in smelt appears to result from a seasonal cycle of GPDH gene expression, with an ensuing increase in the expression of PEPCK, and a similar but independent cycle of AFP gene expression.

  12. Frostbite Protection in Mice Expressing an Antifreeze Glycoprotein

    PubMed Central

    Heisig, Martin; Mattessich, Sarah; Rembisz, Alison; Acar, Ali; Shapiro, Martin; Booth, Carmen J.; Neelakanta, Girish; Fikrig, Erol

    2015-01-01

    Ectotherms in northern latitudes are seasonally exposed to cold temperatures. To improve survival under cold stress, they use diverse mechanisms to increase temperature resistance and prevent tissue damage. The accumulation of anti-freeze proteins that improve cold hardiness occurs in diverse species including plants, arthropods, fish, and amphibians. We previously identified an Ixodes scapularis anti-freeze glycoprotein, named IAFGP, and demonstrated its cold protective function in the natural tick host and in a transgenic Drosophila model. Here we show, in a transgenic mouse model expressing an anti-freeze glycoprotein, that IAFGP protects mammalian cells and mice from cold shock and frostbite respectively. Transgenic skin samples showed reduced cell death upon cold storage ex vivo and transgenic mice demonstrated increased resistance to frostbite injury in vivo. IAFGP actively protects mammalian tissue from freezing, suggesting its application for the prevention of frostbite, and other diseases associated with cold exposure. PMID:25714402

  13. Biomimetic peptoid oligomers as dual-action antifreeze agents

    PubMed Central

    Huang, Mia L.; Ehre, David; Jiang, Qi; Hu, Chunhua; Kirshenbaum, Kent; Ward, Michael D.

    2012-01-01

    The ability of natural peptides and proteins to influence the formation of inorganic crystalline materials has prompted the design of synthetic compounds for the regulation of crystal growth, including the freezing of water and growth of ice crystals. Despite their versatility and ease of structural modification, peptidomimetic oligomers have not yet been explored extensively as crystallization modulators. This report describes a library of synthetic N-substituted glycine peptoid oligomers that possess “dual-action” antifreeze activity as exemplified by ice crystal growth inhibition concomitant with melting temperature reduction. We investigated the structural features responsible for these phenomena and observed that peptoid antifreeze activities depend both on oligomer backbone structure and side chain chemical composition. These studies reveal the capability of peptoids to act as ice crystallization regulators, enabling the discovery of a unique and diverse family of synthetic oligomers with potential as antifreeze agents in food production and biomedicine. PMID:23169638

  14. Expression of lectin-like oxidized LDL receptor-1 in smooth muscle cells after vascular injury

    SciTech Connect

    Eto, Hideyuki; Miyata, Masaaki . E-mail: miyatam@m3.kufm.kagoshima-u.ac.jp; Kume, Noriaki; Minami, Manabu; Itabe, Hiroyuki; Orihara, Koji; Hamasaki, Shuichi; Biro, Sadatoshi; Otsuji, Yutaka; Kita, Toru; Tei, Chuwa

    2006-03-10

    Lectin-like oxidized LDL receptor-1 (LOX-1) is an oxidized LDL receptor, and its role in restenosis after angioplasty remains unknown. We used a balloon-injury model of rabbit aorta, and reverse transcription-polymerase chain reaction revealed that LOX-1 mRNA expression was modest in the non-injured aorta, reached a peak level 2 days after injury, and remained elevated until 24 weeks after injury. Immunohistochemistry and in situ hybridization showed that LOX-1 was not detected in the media of non-injured aorta but expressed in both medial and neointimal smooth muscle cells (SMC) at 2 and 24 weeks after injury. Low concentrations of ox-LDL (10 {mu}g/mL) stimulated the cultured SMC proliferation, which was inhibited by antisense oligonucleotides of LOX-1 mRNA. Double immunofluorescense staining showed the colocalization of LOX-1 and proliferating cell nuclear antigen in human restenotic lesion. These results suggest that LOX-1 mediates ox-LDL-induced SMC proliferation and plays a role in neointimal formation after vascular injury.

  15. Antifreeze poisoning: A case report

    PubMed Central

    Wu, Xiaofei; Lu, Guoyu; Qi, Benquan; Wang, Ran; Guo, Daohua; Liu, Xiaolin

    2017-01-01

    The current study reported the case of a 35-year-old male that presented with antifreeze poisoning. The clinical manifestations, laboratory investigations and treatments were analyzed, and the obtained results were compared with those in previous reports. Subsequent to consuming antifreeze, the patient mainly presented nausea and agitation, without disturbance of consciousness. Laboratory investigations indicated severe metabolic acidosis, renal dysfunction and hyperkalemia. The patient underwent hemodialysis and his condition was significantly improved on the day of admission. Renal function gradually deteriorated, but was eventually improved due to treatment, including hemodialysis, mannitol for catharsis, furosemide for diuresis, Xuebijing for the removal of blood stasis and detoxication, and reduced glutathione for the protection of major organs. The patient was discharged 1 month after hospital admission. In conclusion, the significance and clinical manifestations of antifreeze poisoning should be identified in clinical practice, and active hemodialysis should be provided. The aim of the current study was to summarize the clinical manifestations and treatments of patients with antifreeze poisoning, and to advance the recognition of antifreeze poisoning. PMID:28352354

  16. Lectin-Like Bacteriocins from Pseudomonas spp. Utilise D-Rhamnose Containing Lipopolysaccharide as a Cellular Receptor

    PubMed Central

    Josts, Inokentijs; Roszak, Aleksander W.; Waløen, Kai I.; Cogdell, Richard J.; Milner, Joel; Evans, Tom; Kelly, Sharon; Tucker, Nicholas P.; Byron, Olwyn; Smith, Brian; Walker, Daniel

    2014-01-01

    Lectin-like bacteriocins consist of tandem monocot mannose-binding domains and display a genus-specific killing activity. Here we show that pyocin L1, a novel member of this family from Pseudomonas aeruginosa, targets susceptible strains of this species through recognition of the common polysaccharide antigen (CPA) of P. aeruginosa lipopolysaccharide that is predominantly a homopolymer of d-rhamnose. Structural and biophysical analyses show that recognition of CPA occurs through the C-terminal carbohydrate-binding domain of pyocin L1 and that this interaction is a prerequisite for bactericidal activity. Further to this, we show that the previously described lectin-like bacteriocin putidacin L1 shows a similar carbohydrate-binding specificity, indicating that oligosaccharides containing d-rhamnose and not d-mannose, as was previously thought, are the physiologically relevant ligands for this group of bacteriocins. The widespread inclusion of d-rhamnose in the lipopolysaccharide of members of the genus Pseudomonas explains the unusual genus-specific activity of the lectin-like bacteriocins. PMID:24516380

  17. Lectin-like bacteriocins from Pseudomonas spp. utilise D-rhamnose containing lipopolysaccharide as a cellular receptor.

    PubMed

    McCaughey, Laura C; Grinter, Rhys; Josts, Inokentijs; Roszak, Aleksander W; Waløen, Kai I; Cogdell, Richard J; Milner, Joel; Evans, Tom; Kelly, Sharon; Tucker, Nicholas P; Byron, Olwyn; Smith, Brian; Walker, Daniel

    2014-02-01

    Lectin-like bacteriocins consist of tandem monocot mannose-binding domains and display a genus-specific killing activity. Here we show that pyocin L1, a novel member of this family from Pseudomonas aeruginosa, targets susceptible strains of this species through recognition of the common polysaccharide antigen (CPA) of P. aeruginosa lipopolysaccharide that is predominantly a homopolymer of D-rhamnose. Structural and biophysical analyses show that recognition of CPA occurs through the C-terminal carbohydrate-binding domain of pyocin L1 and that this interaction is a prerequisite for bactericidal activity. Further to this, we show that the previously described lectin-like bacteriocin putidacin L1 shows a similar carbohydrate-binding specificity, indicating that oligosaccharides containing D-rhamnose and not D-mannose, as was previously thought, are the physiologically relevant ligands for this group of bacteriocins. The widespread inclusion of d-rhamnose in the lipopolysaccharide of members of the genus Pseudomonas explains the unusual genus-specific activity of the lectin-like bacteriocins.

  18. The lectin-like oxidized LDL receptor-1: a new potential molecular target in colorectal cancer

    PubMed Central

    Murdocca, Michela; Mango, Ruggiero; Pucci, Sabina; Biocca, Silvia; Testa, Barbara; Capuano, Rosamaria; Paolesse, Roberto; Sanchez, Massimo; Orlandi, Augusto; di Natale, Corrado; Novelli, Giuseppe; Sangiuolo, Federica

    2016-01-01

    The identification of new biomarkers and targets for tailored therapy in human colorectal cancer (CRC) onset and progression is an interesting challenge. CRC tissue produces an excess of ox-LDL, suggesting a close correlation between lipid dysfunction and malignant transformation. Lectin-like oxidized LDL receptor-1 (LOX-1) is involved in several mechanisms closely linked to tumorigenesis. Here we report a tumor specific LOX-1 overexpression in human colon cancers: LOX-1 results strongly increased in the 72% of carcinomas (P<0.001), and strongly overexpressed in 90% of highly aggressive and metastatic tumours (P<0.001), as compared to normal mucosa. Moreover LOX-1 results modulated since the early stage of the disease (adenomas vs normal mucosa; P<0.001) suggesting an involvement in tumor insurgence and progression. The in vitro knockdown of LOX-1 in DLD-1 and HCT-8 colon cancer cells by siRNA and anti-LOX-1 antibody triggers to an impaired proliferation rate and affects the maintenance of cell growth and tumorigenicity. The wound-healing assay reveals an evident impairment in closing the scratch. Lastly knockdown of LOX-1 delineates a specific pattern of volatile compounds characterized by the presence of a butyrate derivative, suggesting a potential role of LOX-1 in tumor-specific epigenetic regulation in neoplastic cells. The role of LOX-1 as a novel biomarker and molecular target represents a concrete opportunity to improve current therapeutic strategies for CRC. In addition, the innovative application of a technology focused to the identification of LOX-1 driven volatiles specific to colorectal cancer provides a promising diagnostic tool for CRC screening and for monitoring the response to therapy. PMID:26895376

  19. Lectin-Like Constituents of Foods Which React with Components of Serum, Saliva, and Streptococcus mutans

    PubMed Central

    Gibbons, R. J.; Dankers, I.

    1981-01-01

    Hot and cold aqueous extracts were prepared from 22 commonly ingested fruits, vegetables, and seeds. When tested by agar diffusion, extracts from 13 and 10 of the foods formed precipitin bands with samples of normal rabbit serum and human saliva, respectively; extracts from four of the foods also reacted with antigen extracts of strains of Streptococcus mutans. When added to rabbit antiserum, extracts from 18 of 21 foods tested inhibited reactivity with antigen extracts derived from S. mutans MT3. Extracts from 16 foods agglutinated whole S. mutans cells, whereas those from 10 foods agglutinated human erythrocytes of blood types A and B. The lectin-like activities of extracts which reacted with human saliva were studied further. Pretreatment of saliva-coated hydroxyapatite (S-HA) beads with extracts of bananas, coconuts, carrots, alfalfa, and sunflower seeds markedly reduced the subsequent adsorption of S. mutans MT3. Pretreatment of S-HA with banana extract also strongly inhibited adsorption of S. mutans H12 and S. sanguis C1, but it had little effect on attachment of Actinomyces naeslundii L13 or A. viscosus LY7. Absorption experiments indicated that the component(s) in banana extract responsible for inhibiting streptococcal adsorption to S-HA was identical to that which bound to human erythrocytes. The banana hemagglutinin exhibited highest activity between pH 7 and 8, and it was inhibited by high concentrations of glucosamine, galactosamine, and, to a lesser extent, mannosamine. Other sugars tested had no effect. The selective bacterial adsorption-inhibiting effect noted for banana extract was also observed in studies with purified lectins. Thus, pretreating S-HA with wheat germ agglutinin and concanavalin A inhibited adsorption of S. mutans MT3 cells, whereas peanut agglutinin, Ulex agglutinin, Dolichos agglutinin, and soybean agglutinin had little effect; none of these lectins affected attachment of A. viscosus LY7. Collectively, the observations suggest that

  20. Nontoxic Antifreeze for Insect Traps

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Propylene glycol in water is a safe and effective alternative to ethylene glycol as a capture liquid in insect traps (pitfalls, flight intercepts, pan traps). Propylene glycol formulations are readily available because it is the primary (95%) ingredient in certain automotive antifreeze formulations...

  1. Laboratory Evaluation of Commercial Antifreezes.

    DTIC Science & Technology

    1986-09-01

    accomplished by atomic absorption spectroscopy . No attempt \\%as made to identify minor components. Conley, James H.’and Jamison, Robert G.. "Evaluation...freeze point depressant. Analysis by atomic absorption spectroscopy shows that all products, except Antifreeze G, contain boron. All II commercial

  2. C-type lectin-like molecule-1 (CLL1)-targeted TRAIL augments the tumoricidal activity of granulocytes and potentiates therapeutic antibody-dependent cell-mediated cytotoxicity.

    PubMed

    Wiersma, Valerie R; de Bruyn, Marco; Shi, Ce; Gooden, Marloes J M; Wouters, Maartje C A; Samplonius, Douwe F; Hendriks, Djoke; Nijman, Hans W; Wei, Yunwei; Zhou, Jin; Helfrich, Wijnand; Bremer, Edwin

    2015-01-01

    The therapeutic effect of anti-cancer monoclonal antibodies stems from their capacity to opsonize targeted cancer cells with subsequent phagocytic removal, induction of antibody-dependent cell-mediated cytotoxicity (ADCC) or induction of complement-mediated cytotoxicity (CDC). The major immune effector cells involved in these processes are natural killer (NK) cells and granulocytes. The latter and most prevalent blood cell population contributes to phagocytosis, but is not effective in inducing ADCC. Here, we report that targeted delivery of the tumoricidal protein tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to granulocyte marker C-type lectin-like molecule-1 (CLL1), using fusion protein CLL1:TRAIL, equips granulocytes with high levels of TRAIL. Upon CLL1-selective binding of this fusion protein, granulocytes acquire additional TRAIL-mediated cytotoxic activity that, importantly, potentiates antibody-mediated cytotoxicity of clinically used therapeutic antibodies (e.g., rituximab, cetuximab). Thus, CLL1:TRAIL could be used as an adjuvant to optimize the clinical potential of anticancer antibody therapy by augmenting tumoricidal activity of granulocytes.

  3. Expression, purification and characterization of a group of lectin-like peptides from the spider Ornithoctonus huwena.

    PubMed

    Jiang, Liping; Peng, Li; Zhang, Yongqun; Chen, Jinjun; Zhang, Dongyi; Liang, Songping

    2009-04-01

    By sequencing random clones from the venom gland cDNA library of the spider Ornithoctonus huwena, a transcript, named SHL-Ib1, encoding a lectin-like peptide was cloned. The amino acid sequence of the putative mature peptide of SHL-Ib1 is identical, except for seven different residues, with that of SHL-I, a lectin found in the venom of O. huwena. The mature peptides of SHL-Ib1b and SHL-Ib1c are the mutants of SHL-Ib1 with two or three amino acid residues truncated at the C-terminal. The recombinant SHL-Ib1b and SHL-Ib1c were expressed successfully by the yeast expression system and purified by using a combination of ion-exchange and reverse phase high performance liquid chromatography (HPLC). The molecular masses of the two expressed peptides were identified by mass spectrometry, indicating that the C-terminals of the two peptides were not amidated. The two peptides can agglutinate human erythrocytes at minimal concentrations of 0.75 and 1.475mg/ml, respectively. Structure modeling of SHL-Ib1 has given a clue to the low agglutination bioactivities of these recombinant toxins. These lectin-like peptides, due to the small molecular sizes, may have the advantage to investigate the binding mechanism of the lectin and have the potential to be the carrier for drug delivery.

  4. Serum amyloid A stimulates macrophage foam cell formation via lectin-like oxidized low-density lipoprotein receptor 1 upregulation

    SciTech Connect

    Lee, Ha Young; Kim, Sang Doo; Baek, Suk-Hwan; Choi, Joon Hyuk; Cho, Kyung-Hyun; Zabel, Brian A.; Bae, Yoe-Sik

    2013-03-29

    Highlights: ► SAA induced macrophage foam cell formation. ► SAA stimulated upregulation of lectin-like oxidized low-density lipoprotein receptor 1 (LOX1). ► SAA-induced LOX1 expression and foam cell formation is mediated by JNK/NF-κB signaling. ► HDL-conjugated SAA also stimulates foam cell formation via LOX1 upregulation. ► The finding reveals a novel mechanism of action of SAA in the pathogenesis of atherosclerosis. -- Abstract: Elevated levels of serum amyloid A (SAA) is a risk factor for cardiovascular diseases, however, the role of SAA in the pathophysiology of atherosclerosis remains unclear. Here we show that SAA induced macrophage foam cell formation. SAA-stimulated foam cell formation was mediated by c-jun N-terminal kinase (JNK) signaling. Moreover, both SAA and SAA-conjugated high density lipoprotein stimulated the expression of the important scavenger receptor lectin-like oxidized low-density lipoprotein receptor 1 (LOX1) via nuclear factor-κB (NF-κB). A LOX1 antagonist carrageenan significantly blocked SAA-induced foam cell formation, indicating that SAA promotes foam cell formation via LOX1 expression. Our findings therefore suggest that SAA stimulates foam cell formation via LOX1 induction, and thus likely contributes to atherogenesis.

  5. Viable head and neck tumor spheroids stimulate in vitro autologous monocyte MCP-1 secretion through soluble substances and CD14/lectin-like receptors.

    PubMed

    Olsnes, Carla; Heimdal, John-Helge; Kross, Kenneth W; Olofsson, Jan; Aarstad, Hans Jørgen

    2005-12-01

    Biopsies from carcinoma tissue and benign control mucosa from head and neck squamous cell carcinoma (HNSCC) patients were used to establish fragment (F)-spheroids in vitro. We have previously shown that autologous monocytes co-cultured with F-spheroids in vitro augment their secretion of monocyte chemotactic protein-1 (MCP-1). Presently, the aims of the present work were to study whether the metabolic activity, secreted products and/or specific receptor/ligand on the surface of the F-spheroids and monocytes are necessary for stimulation of the monocyte MCP-1 secretion upon F-spheroid co-culture. Actinomycin D (1 mug/ml for 24 h) pre-treatment of the F-spheroids abolished the monocyte MCP-1 co-culture response. Co-culture of monocytes and F-spheroids separated by a semi-permeable membrane showed a decreased, but still present, monocyte MCP-1 co-culture response. Conditioned medium from F-spheroids stimulated allogenous monocytes to secrete MCP-1. The addition of glucose or galactose, but not mannose, to co-cultures partially inhibited the monocyte MCP-1 co-culture response. The addition of anti-CD14 antibody diminished the MCP-1 co-culture response. In conclusion, the monocyte MCP-1 co-culture response is dependent on metabolically active spheroids, secreted stimuli, and is augmented by direct contact with F-spheroids, possibly via lectin-like receptors and the CD14 receptor.

  6. C-type lectin-like receptor LOX-1 promotes dendritic cell-mediated class-switched B cell responses.

    PubMed

    Joo, HyeMee; Li, Dapeng; Dullaers, Melissa; Kim, Tae-Whan; Duluc, Dorothee; Upchurch, Katherine; Xue, Yaming; Zurawski, Sandy; Le Grand, Roger; Liu, Yong-Jun; Kuroda, Marcelo; Zurawski, Gerard; Oh, SangKon

    2014-10-16

    Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a pattern-recognition receptor for a variety of endogenous and exogenous ligands. However, LOX-1 function in the host immune response is not fully understood. Here, we report that LOX-1 expressed on dendritic cells (DCs) and B cells promotes humoral responses. On B cells LOX-1 signaling upregulated CCR7, promoting cellular migration toward lymphoid tissues. LOX-1 signaling on DCs licensed the cells to promote B cell differentiation into class-switched plasmablasts and led to downregulation of chemokine receptor CXCR5 and upregulation of chemokine receptor CCR10 on plasmablasts, enabling their exit from germinal centers and migration toward local mucosa and skin. Finally, we found that targeting influenza hemagglutinin 1 (HA1) subunit to LOX-1 elicited HA1-specific protective antibody responses in rhesus macaques. Thus, LOX-1 expressed on B cells and DC cells has complementary functions to promote humoral immune responses.

  7. Structure and collective dynamics of hydrated anti-freeze protein type III from 180 K to 298 K by X-ray diffraction and inelastic X-ray scattering

    NASA Astrophysics Data System (ADS)

    Yoshida, Koji; Baron, Alfred Q. R.; Uchiyama, Hiroshi; Tsutsui, Satoshi; Yamaguchi, Toshio

    2016-04-01

    We investigated hydrated antifreeze protein type III (AFP III) powder with a hydration level h (=mass of water/mass of protein) of 0.4 in the temperature range between 180 K and 298 K using X-ray diffraction and inelastic X-ray scattering (IXS). The X-ray diffraction data showed smooth, largely monotonic changes between 180 K and 298 K without freezing water. Meanwhile, the collective dynamics observed by IXS showed a strong change in the sound velocity at 180 K, after being largely temperature independent at higher temperatures (298-220 K). We interpret this change in terms of the dynamic transition previously discussed using other probes including THz IR absorption spectroscopy and incoherent elastic and quasi-elastic neutron scattering. This finding suggests that the dynamic transition of hydrated proteins is observable on the subpicosecond time scale as well as nano- and pico-second scales, both in collective dynamics from IXS and single particle dynamics from neutron scattering. Moreover, it is most likely that the dynamic transition of hydrated AFP III is not directly correlated with its hydration structure.

  8. Arsenic augments the uptake of oxidized LDL by upregulating the expression of lectin-like oxidized LDL receptor in mouse aortic endothelial cells

    SciTech Connect

    Hossain, Ekhtear; Ota, Akinobu; Karnan, Sivasundaram; Damdindorj, Lkhagvasuren; Takahashi, Miyuki; Konishi, Yuko; Konishi, Hiroyuki; Hosokawa, Yoshitaka

    2013-12-15

    Although chronic arsenic exposure is a well-known risk factor for cardiovascular diseases, including atherosclerosis, the molecular mechanism underlying arsenic-induced atherosclerosis remains obscure. Therefore, this study aimed to elucidate this molecular mechanism. We examined changes in the mRNA level of the lectin-like oxidized LDL (oxLDL) receptor (LOX-1) in a mouse aortic endothelial cell line, END-D, after sodium arsenite (SA) treatment. SA treatment significantly upregulated LOX-1 mRNA expression; this finding was also verified at the protein expression level. Flow cytometry and fluorescence microscopy analyses showed that the cellular uptake of fluorescence (Dil)-labeled oxLDL was significantly augmented with SA treatment. In addition, an anti-LOX-1 antibody completely abrogated the augmented uptake of Dil-oxLDL. We observed that SA increased the levels of the phosphorylated forms of nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB)/p65. SA-induced upregulation of LOX-1 protein expression was clearly prevented by treatment with an antioxidant, N-acetylcysteine (NAC), or an NF-κB inhibitor, caffeic acid phenethylester (CAPE). Furthermore, SA-augmented uptake of Dil-oxLDL was also prevented by treatment with NAC or CAPE. Taken together, our results indicate that arsenic upregulates LOX-1 expression through the reactive oxygen species-mediated NF-κB signaling pathway, followed by augmented cellular oxLDL uptake, thus highlighting a critical role of the aberrant LOX-1 signaling pathway in the pathogenesis of arsenic-induced atherosclerosis. - Highlights: • Sodium arsenite (SA) increases LOX-1 expression in mouse aortic endothelial cells. • SA enhances cellular uptake of oxidized LDL in dose-dependent manner. • SA-induced ROS generation enhances phosphorylation of NF-κB. • SA upregulates LOX-1 expression through ROS-activated NF-κB signaling pathway.

  9. Refolding of β-Stranded Class I Chitinases of Hippophae rhamnoides Enhances the Antifreeze Activity during Cold Acclimation

    PubMed Central

    Gupta, Ravi; Deswal, Renu

    2014-01-01

    Class I chitinases hydrolyse the β-1,4-linkage of chitin and also acquire antifreeze activity in some of the overwintering plants during cold stress. Two chitinases, HrCHT1a of 31 kDa and HrCHT1b of 34 kDa, were purified from cold acclimated and non-acclimated seabuckthorn seedlings using chitin affinity chromatography. 2-D gels of HrCHT1a and HrCHT1b showed single spots with pIs 7.0 and 4.6 respectively. N-terminal sequence of HrCHT1b matched with the class I chitinase of rice and antifreeze proteins while HrCHT1a could not be sequenced as it was N-terminally blocked. Unlike previous reports, where antifreeze activity of chitinase was cold inducible, our results showed that antifreeze activity is constitutive property of class I chitinase as both HrCHT1a and HrCHT1b isolated even from non-acclimated seedlings, exhibited antifreeze activity. Interestingly, HrCHT1a and HrCHT1b purified from cold acclimated seedlings, exhibited 4 and 2 times higher antifreeze activities than those purified from non-acclimated seedlings, suggesting that antifreeze activity increased during cold acclimation. HrCHT1b exhibited 23–33% higher hydrolytic activity and 2–4 times lower antifreeze activity than HrCHT1a did. HrCHT1b was found to be a glycoprotein; however, its antifreeze activity was independent of glycosylation as even deglycosylated HrCHT1b exhibited antifreeze activity. Circular dichroism (CD) analysis showed that both these chitinases were rich in unusual β-stranded conformation (36–43%) and the content of β-strand increased (∼11%) during cold acclimation. Surprisingly, calcium decreased both the activities of HrCHT1b while in case of HrCHT1a, a decrease in the hydrolytic activity and enhancement in its antifreeze activity was observed. CD results showed that addition of calcium also increased the β-stranded conformation of HrCHT1a and HrCHT1b. This is the first report, which shows that antifreeze activity is constitutive property of class I chitinase and cold

  10. Novel Lectin-Like Bacteriocins of Biocontrol Strain Pseudomonas fluorescens Pf-5

    PubMed Central

    Parret, Annabel H. A.; Temmerman, Koen; De Mot, René

    2005-01-01

    Bacteriocin LlpA, produced by Pseudomonas sp. strain BW11M1, is a peculiar antibacterial protein due to its homology to mannose-binding lectins mostly found in monocots (A. H. A. Parret, G. Schoofs, P. Proost, and R. De Mot, J. Bacteriol. 185:897-908, 2003). Biocontrol strain Pseudomonas fluorescens Pf-5 contains two llpA-like genes, named llpA1Pf-5 and llpA2Pf-5. Recombinant Escherichia coli cells expressing llpA1Pf-5 or llpA2Pf-5 acquired bacteriocin activity and secreted a 31-kDa protein cross-reacting with LlpABW11M1 antibodies. Antibacterial activity of the recombinant proteins was evidenced by gel overlay assays. Analysis of the antimicrobial spectrum indicated that LlpA1Pf-5 and LlpA2Pf-5 are able to inhibit P. fluorescens strains, as well as the related mushroom pathogen Pseudomonas tolaasii. LlpA-type bacteriocins are characterized by a domain structure consisting of tandem monocot mannose-binding lectin (MMBL) domains. Molecular phylogeny of these MMBL domains suggests that the individual MMBL domains within an LlpA protein have evolved separately toward a specific, as yet unknown, function or, alternatively, were acquired from different ancestral sources. Our observations are consistent with earlier observations, which hinted that MMBL-like bacteriocins represent a new family of antibacterial proteins, probably with a novel mode of action. PMID:16151105

  11. Taurine suppresses oxidative stress-potentiated expression of lectin-like oxidized low-density lipoprotein receptor and restenosis in balloon-injured rabbit iliac artery.

    PubMed

    Gokce, G; Ozsarlak-Sozer, G; Oran, I; Oktay, G; Ozkal, S; Kerry, Z

    2011-12-01

    1. In endothelial cells, the major receptor for the binding and internalization of oxidized low-density lipoprotein (LDL) is the lectin-like oxidized LDL receptor (LOX-1). The aim of the present study was to investigate the effects of taurine on intimal thickening and LOX-1 expression under normal and oxidative conditions. 2. The iliac artery of rabbits were subjected to balloon injury and oxidative stress was induced by 14 days treatment of rabbits with 75 mg/kg, s.c., buthionine sulfoximine (BSO), a specific inhibitor of glutathione synthesis. Taurine was administered in drinking water (1%, w/v) for 14 days in the presence (BSO + Taurine group) and in the absence of BSO treatment (Taurine group). In taurine and placebo groups, rabbits were injected with 4 mL, s.c., 0.9% NaCl (vehicle for BSO) for 14 days. 3. Taurine (1% in drinking water, w/v) preserved plasma levels of anti-oxidants and lowered the increased blood pressure induced by BSO. The stenosis rate of 29.92% in the placebo group increased to 72.20% in the BSO group, which was significantly reduced to 42.21% by taurine (P < 0.001; n = 5). Localization of LOX-1 to the intima and media of the iliac artery was demonstrated in the present study. Taurine treatment reduced the BSO-induced increase in LOX-1 expression at both the protein and mRNA levels (P < 0.05 and P < 0.01, respectively). 4. The results demonstrate that the stenosis rate and LOX-1 expression correlate well with oxidative status. Manipulation of LOX-1 expression by taurine may have therapeutic benefits in preventing restenosis.

  12. Lectin-like oxidized LDL receptor-1 is an enhancer of tumor angiogenesis in human prostate cancer cells.

    PubMed

    González-Chavarría, Iván; Cerro, Rita P; Parra, Natalie P; Sandoval, Felipe A; Zuñiga, Felipe A; Omazábal, Valeska A; Lamperti, Liliana I; Jiménez, Silvana P; Fernandez, Edelmira A; Gutiérrez, Nicolas A; Rodriguez, Federico S; Onate, Sergio A; Sánchez, Oliberto; Vera, Juan C; Toledo, Jorge R

    2014-01-01

    Altered expression and function of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) has been associated with several diseases such as endothelial dysfunction, atherosclerosis and obesity. In these pathologies, oxLDL/LOX-1 activates signaling pathways that promote cell proliferation, cell motility and angiogenesis. Recent studies have indicated that olr1 mRNA is over-expressed in stage III and IV of human prostatic adenocarcinomas. However, the function of LOX-1 in prostate cancer angiogenesis remains to be determined. Our aim was to analyze the contribution of oxLDL and LOX-1 to tumor angiogenesis using C4-2 prostate cancer cells. We analyzed the expression of pro-angiogenic molecules and angiogenesis on prostate cancer tumor xenografts, using prostate cancer cell models with overexpression or knockdown of LOX-1 receptor. Our results demonstrate that the activation of LOX-1 using oxLDL increases cell proliferation, and the expression of the pro-angiogenic molecules VEGF, MMP-2, and MMP-9 in a dose-dependent manner. Noticeably, these effects were prevented in the C4-2 prostate cancer model when LOX-1 expression was knocked down. The angiogenic effect of LOX-1 activated with oxLDL was further demonstrated using the aortic ring assay and the xenograft model of tumor growth on chorioallantoic membrane of chicken embryos. Consequently, we propose that LOX-1 activation by oxLDL is an important event that enhances tumor angiogenesis in human prostate cancer cells.

  13. Identification of lectin-like substances recognizing galactosyl residues of glycoconjugates on the plasma membrane of marrow sinus endothelium

    SciTech Connect

    Kataoka, M.; Tavassoli, M.

    1985-05-01

    Plasma membrane components capable of specific binding to the sugar residues of glycoproteins have recently been identified in several cell systems and implicated in the recognition and specific uptake of soluble or membrane-bound glycoproteins. Because the endothelium of bone marrow sinuses is the site of massive cellular and molecular traffic that is often specific, we studied the surface of sinus endothelium for the presence of sugar-recognizing systems. Neoglycoprotein probes were synthesized by covalently binding bovine serum albumin (BSA) to activated pyrannose form of galactose, fucose, or mannose. The probe was then labeled with colloidal gold. Galactosyl-BSA gold bound to endothelial membrane at 4 degrees C and was internalized at 37 degrees C. Both the binding and the internalization were inhibited in the presence of excess unlabeled galactosyl-BSA. Gold-labeled mannosyl-BSA and fucosyl-BSA did not bind to the endothelium. Nor did BSA-gold without galactosyl residues bind to endothelial membrane, confirming that galactosyl moiety was responsible for the binding. The uptake of galactosyl-BSA was further confirmed by perfusion of /sup 125/I-galactosyl-BSA into the abdominal aorta. Small but highly specific uptake was noted in tibias and femurs. These data provide evidence for the presence of a lectin-like substance on the luminal surface of marrow endothelial membrane capable of specific interaction with galactosyl residues of circulating and cell-bound glycoproteins and may provide a mechanism for specific recognition of these glycoproteins.

  14. Lectin-like oxidized LDL receptor-1 expresses in mouse bone marrow-derived mesenchymal stem cells and stimulates their proliferation

    SciTech Connect

    Zhang, Fenxi; Wang, Congrui; Jing, Suhua; Ren, Tongming; Li, Yonghai; Cao, Yulin; Lin, Juntang

    2013-04-15

    The bone marrow-derived mesenchymal stem cells (bmMSCs) have been widely used in cell transplant therapy, and the proliferative ability of bmMSCs is one of the determinants of the therapy efficiency. Lectin-like oxidized low density lipoprotein receptor-1 (LOX-1) as a transmembrane protein is responsible for binding, internalizing and degrading oxidized low density lipoprotein (ox-LDL). It has been identified that LOX-1 is expressed in endothelial cells, vascular smooth muscle cells, cardiomyocytes, fibroblasts and monocytes. In these cells, low concentration of ox-LDL (<40 μg/mL) stimulates their proliferation via LOX-1 activation. However, it is poor understood that whether LOX-1 is expressed in bmMSCs and which role it plays. In this study, we investigated the status of LOX-1 expression in bmMSCs and its function on bmMSC proliferation. Our results showed that primary bmMSCs exhibiting a typical fibroblast-like morphology are positive for CD44 and CD90, but negative for CD34 and CD45. LOX-1 in both mRNA and protein levels is highly expressed in bmMSCs. Meanwhile, bmMSCs exhibit a strong potential to take up ox-LDL. Moreover, LOX-1 expression in bmMSCs is upregulated by ox-LDL with a dose- and time-dependent manner. Presence of ox-LDL also enhances the proliferation of bmMSCs. Knockdown of LOX-1 expression significantly inhibits ox-LDL-induced bmMSC proliferation. These findings indicate that LOX-1 plays a role in bmMSC proliferation. - Highlights: ► LOX-1 expresses in bmMSCs and mediates uptake of ox-LDL. ► Ox-LDL stimulates upregulation of LOX-1 in bmMSCs. ► Ox-LDL promotes bmMSC proliferation and expression of Mdm2, phosphor-Akt, phosphor-ERK1/2 and phosphor-NF-κB. ► LOX-1 siRNA inhibits ox-LDL-induced bmMSC proliferation and expression cell survival signals.

  15. A nonprotein thermal hysteresis-producing xylomannan antifreeze in the freeze-tolerant Alaskan beetle Upis ceramboides

    PubMed Central

    Walters, Kent R.; Serianni, Anthony S.; Sformo, Todd; Barnes, Brian M.; Duman, John G.

    2009-01-01

    Thermal hysteresis (TH), a difference between the melting and freezing points of a solution that is indicative of the presence of large-molecular-mass antifreezes (e.g., antifreeze proteins), has been described in animals, plants, bacteria, and fungi. Although all previously described TH-producing biomolecules are proteins, most thermal hysteresis factors (THFs) have not yet been structurally characterized, and none have been characterized from a freeze-tolerant animal. We isolated a highly active THF from the freeze-tolerant beetle, Upis ceramboides, by means of ice affinity. Amino acid chromatographic analysis, polyacrylamide gel electrophoresis, UV-Vis spectrophotometry, and NMR spectroscopy indicated that the THF contained little or no protein, yet it produced 3.7 ± 0.3 °C of TH at 5 mg/ml, comparable to that of the most active insect antifreeze proteins. Compositional and structural analyses indicated that this antifreeze contains a β-mannopyranosyl-(1→4) β-xylopyranose backbone and a fatty acid component, although the lipid may not be covalently linked to the saccharide. Consistent with the proposed structure, treatment with endo-β-(1→4)xylanase ablated TH activity. This xylomannan is the first TH-producing antifreeze isolated from a freeze-tolerant animal and the first in a new class of highly active THFs that contain little or no protein. PMID:19934038

  16. Threonine side chain conformational population distribution of a type I antifreeze protein on interacting with ice surface studied via ¹³C-¹⁵N dynamic REDOR NMR.

    PubMed

    Mao, Yougang; Jeong, Myongho; Wang, Tieli; Ba, Yong

    2011-01-01

    Antifreeze proteins (AFPs) provide survival mechanism for species living in subzero environments by lowering the freezing points of their body fluids effectively. The mechanism is attributed to AFPs' ability to inhibit the growth of seed ice crystals through adsorption on specific ice surfaces. We have applied dynamic REDOR (Rotational Echo Double Resonance) solid state NMR to study the threonine (Thr) side chain conformational population distribution of a site-specific Thr ¹³C(γ) and ¹⁵N doubly labeled type I AFP in frozen aqueous solution. It is known that the Thr side chains together with those of the 4th and 8th Alanine (Ala) residues commencing from the Thrs (the 1st) in the four 11-residue repeat units form the peptide ice-binding surface. The conformational information can provide structural insight with regard to how the AFP side chains structurally interact with the ice surface. χ-squared statistical analysis of the experimental REDOR data in fitting the theoretically calculated dynamic REDOR fraction curves indicates that when the AFP interacted with the ice surface in the frozen AFP solution, the conformations of the Thr side chains changed from the anti-conformations, as in the AFP crystal structure, to partial population in the anti-conformation and partial population in the two gauche conformations. This change together with the structural analysis indicates that the simultaneous interactions of the methyl groups and the hydroxyl groups of the Thr side chains with the ice surface could be the reason for the conformational population change. The analysis of the theoretical dynamic REDOR fraction curves shows that the set of experimental REDOR data may fit a number of theoretical curves with different population distributions. Thus, other structural information is needed to assist in determining the conformational population distribution of the Thr side chains.

  17. Lectin-like, oxidized low-density lipoprotein receptor-1-deficient mice show resistance to age-related knee osteoarthritis

    PubMed Central

    Hashimoto, Kazuhiko; Oda, Yutaka; Nakamura, Fumihisa; Kakinoki, Ryosuke; Akagi, Masao

    2017-01-01

    The lectin-like, oxidized low-density lipoprotein (ox-LDL) receptor-1 (LOX-1)/ox-LDL system contributes to atherosclerosis and may be involved in cartilage degeneration. The purpose of this study was to determine whether the LOX-1/ox-LDL system contributes to age-related osteoarthritis (OA) in vivo, using LOX-1 knockout (LOX-1 KO) mice. Knee cartilage from 6, 12, and 18-month old (n = 10/group) C57Bl/6 wild-type (WT) and LOX-1 KO mice was evaluated by determining the Osteoarthritis Research Society International (OARSI) score of Safranin-O stained samples. The prevalence of knee OA in both mouse strains was also investigated. Expression levels of LOX-1, ox-LDL, runt-related transcription factor-2 (Runx2), type-X collagen (COL X), and matrix metalloproteinase-13 (MMP-13) in the articular chondrocytes were analyzed immunohistologically. No significant difference was observed in the mean scores of WT (2.00±0.61) and LOX-1 KO mice (2.00±0.49) at 6 months of age (P=1.00, n=10). At 12 and 18 months of age, the mean scores of LOX-1 KO mice (3.75±0.93 and 5.50±0.78) were significantly lower than those of WT mice (5.25±1.14 and 9.00±1.01; P<0.001 in both cases; n=10). The prevalence of OA in LOX-1 KO mice was lower than that in WT mice at 12 and 18 months of age (40 vs 70%, 70 vs 90%, respectively; n=10). The expression levels of Runx2, COL X, and MMP-13 in articular chondrocytes significantly decreased in LOX-1 KO, mice compared with those in WT mice. The study indicated that the LOX-1/ox-LDL system in chondrocytes plays a role in the pathogenesis of age-related knee OA, which is potentially a target for preventing OA progression. PMID:28348422

  18. Modified Antifreeze Liquids for Use on Surfaces

    NASA Technical Reports Server (NTRS)

    Lynn, R. O.

    1983-01-01

    Report presents results of evaluation of two antifreeze liquids, dimethyl sulfoxide and ethylene glycol and five viscosity modifiers: gelatin, gum tragacanth, starch, agarose powder and citrus pectin. Purpose of evaluation to find best way of dealing with frost formation on Space Shuttle.

  19. Variation in blood serum antifreeze activity of Antarctic Trematomus fishes across habitat temperature and depth.

    PubMed

    Fields, Lauren G; DeVries, Arthur L

    2015-07-01

    High latitude waters in the Southern Ocean can be near their freezing point and remain ice-covered throughout the year whereas lower latitude Southern Ocean waters have seasonal ice coverage and comparatively large (6 °C) annual temperature changes. The genus Trematomus (suborder Notothenioidei) is regarded primarily as a high latitude group because of its abundance there, they also inhabit the warmer regions in smaller numbers. Freeze avoidance in the notothenioids is linked to the presence of two antifreeze proteins (AFPs); the antifreeze glycoproteins (AFGPs) and antifreeze potentiating protein (AFPP), both of which adsorb to internal ice crystals inhibiting growth. Both high and low latitude trematomids possess sufficient AFP to lower their blood freezing point below that of seawater (-1.9 °C). We investigated the contributions of AFGPs and AFPP to the blood freezing point depression to determine how they varied with depth, water temperature, and the presence of ice. High latitude trematomids had lower blood freezing points than those inhabiting lower latitude waters indicating differences in their freeze avoidance capacities. Lower freezing points were associated with higher levels of antifreeze activity due to higher levels of both AFGP and AFPP. Populations of Trematomus hansoni and Trematomus bernacchii from shallow depths appear more freeze avoidant than populations inhabiting deep, ice-free water based on their lower freezing points and higher antifreeze activities. Gel electrophoresis of the trichloroacetic acid-soluble AFGPs indicates that only high molecular weight isoforms, which contribute more to AFGP activity, vary across species as well as between individuals of a species.

  20. Clr-a: A Novel Immune-Related C-Type Lectin-like Molecule Exclusively Expressed by Mouse Gut Epithelium.

    PubMed

    Rutkowski, Emilia; Leibelt, Stefan; Born, Christina; Friede, Miriam E; Bauer, Stefan; Weil, Sandra; Koch, Joachim; Steinle, Alexander

    2017-01-15

    The mouse gut epithelium represents a constitutively challenged environment keeping intestinal commensal microbiota at bay and defending against invading enteric pathogens. The complex immunoregulatory network of the epithelial barrier surveillance also involves NK gene complex (NKC)-encoded C-type lectin-like molecules such as NKG2D and Nkrp1 receptors. To our knowledge, in this study, we report the first characterization of the orphan C-type lectin-like molecule Clr-a encoded by the Clec2e gene in the mouse NKC. Screening of a panel of mouse tissues revealed that Clec2e transcripts are restricted to the gastrointestinal tract. Using Clr-a-specific mAb, we characterize Clr-a as a disulfide-linked homodimeric cell surface glycoprotein. Of note, a substantial fraction of Clr-a molecules are retained intracellularly, and analyses of Clr-a/Clr-f hybrids attribute intracellular retention to both the stalk region and parts of the cytoplasmic domain. Combining quantitative PCR analyses with immunofluorescence studies revealed exclusive expression of Clr-a by intestinal epithelial cells and crypt cells throughout the gut. Challenge with polyinosinic-polycytidylic acid results in a rapid and strong downregulation of intestinal Clr-a expression in contrast to the upregulation of Clr-f, a close relative of Clr-a, that also is specifically expressed by the intestinal epithelium and acts as a ligand of the inhibitory Nkrp1g receptor. Collectively, we characterize expression of the mouse NKC-encoded glycoprotein Clr-a as strictly associated with mouse intestinal epithelium. Downregulation upon polyinosinic-polycytidylic acid challenge and expression by crypt cells clearly distinguish Clr-a from the likewise intestinal epithelium-restricted Clr-f, pointing to a nonredundant function of these highly related C-type lectin-like molecules in the context of intestinal immunosurveillance.

  1. Artificial antifreeze polypeptides: alpha-helical peptides with KAAK motifs have antifreeze and ice crystal morphology modifying properties.

    PubMed

    Zhang, W; Laursen, R A

    1999-07-23

    Antifreeze polypeptides from fish are generally thought to inhibit ice crystal growth by specific adsorption onto ice surfaces and preventing addition of water molecules to the ice lattice. Recent studies have suggested that this adsorption results from hydrogen bonding through the side chains of polar amino acids as well as hydrophobic interactions between the non-polar domains on the ice-binding side of antifreeze polypeptides and the clathrate-like surfaces of ice. In order to better understand the activity of one of the antifreeze polypeptide families, namely the alpha-helical type I antifreeze polypeptides, four alpha-helical peptides having sequences not directly analogous to those of known antifreeze polypeptides and containing only positively charged and non-polar side chains were synthesized. Two peptides with regularly spaced lysine residues, GAAKAAKAAAAAAAKAAKAAAAAAAKAAKAAGGY-NH2 and GAALKAAKAAAAAALKAAKAAAAAALKAAKAAGGY-NH2, showed antifreeze activity, albeit weaker than seen in natural antifreeze polypeptides, by the criteria of freezing point depression (thermal hysteresis) and ice crystal modification to a hexagonal trapezohedron. Peptides with irregular spacing of Lys residues were completely inactive. Up to now, lysine residues have not been generally associated with antifreeze activity, though they have been implicated in some antifreeze polypeptides. This work also shows that lysine residues in themselves, when properly positioned on an alpha-helical polyalanine scaffold, have all the requisite properties needed for such an activity.

  2. Lectin-like oxidized low-density lipoprotein receptor-1 facilitates metastasis of gastric cancer through driving epithelial-mesenchymal transition and PI3K/Akt/GSK3β activation

    PubMed Central

    Li, Can; Zhang, Jie; Wu, Hao; Li, Lili; Yang, Caiting; Song, Shushu; Peng, Peike; Shao, Miaomiao; Zhang, Mingming; Zhao, Junjie; Zhao, Ran; Wu, Weicheng; Ruan, Yuanyuan; Wang, Lan; Gu, Jianxin

    2017-01-01

    Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a pattern recognition receptor that plays a critical role in vascular diseases and host immune response. Recently, our research discovered that LOX-1 could facilitate the uptake of dying cells and cross-presentation of cellular antigen via binding with heat shock proteins, which have a close relationship with gastric neoplasia. Therefore, we speculated that LOX-1 may serve as an oncogene in gastric cancer (GC) development and progression. In this study, through immunohistochemistry staining assay and cancer-related databases, we found that LOX-1 expression was up-regulated in GC tissues and correlated with a poor prognosis in GC patients. The expression of LOX-1 was an independent prognostic factor for OS in GC patients, and the incorporation of LOX-1 with TNM stage is more accurate for predicting prognosis. Additionally, in vitro study by transwell assay and western blot analysis confirmed that LOX-1 could promote the migration and invasion of GC cells by driving epithelial-mesenchymal transition and PI3K/Akt/GSK3β activation. Taken together, we first explored the expression profiles, clinical significance and biological function of LOX-1 in GC, and these data suggest that LOX-1 may represent a promising prognostic biomarker for GC and offer a novel molecular target for GC therapies. PMID:28345638

  3. C-type lectin-like domain and fibronectin-like type II domain of phospholipase A(2) receptor 1 modulate binding and migratory responses to collagen.

    PubMed

    Takahashi, Soichiro; Watanabe, Kazuhiro; Watanabe, Yosuke; Fujioka, Daisuke; Nakamura, Takamitsu; Nakamura, Kazuto; Obata, Jun-ei; Kugiyama, Kiyotaka

    2015-03-24

    Phospholipase A2 receptor 1 (PLA2R) mediates collagen-dependent migration. The mechanisms by which PLA2R interacts with collagen remain unclear. We produced HEK293 cells expressing full-length wild-type PLA2R or a truncated PLA2R that lacks fibronectin-like type II (FNII) domains or several regions of C-type lectin-like domain (CTLD). We show that the CTLD1-2 as well as the FNII domain of PLA2R are responsible for binding to collagen and for collagen-dependent migration. Thus, multiple regions and domains of the extracellular portion of PLA2R participate in the responses to collagen. These data suggest a potentially new mechanism for PLA2R-mediated biological response beyond that of a receptor for secretory PLA2.

  4. Expression of Ixodes scapularis Antifreeze Glycoprotein Enhances Cold Tolerance in Drosophila melanogaster

    PubMed Central

    Neelakanta, Girish; Hudson, Andrew M.; Sultana, Hameeda; Cooley, Lynn; Fikrig, Erol

    2012-01-01

    Drosophila melanogaster experience cold shock injury and die when exposed to low non-freezing temperatures. In this study, we generated transgenic D. melanogaster that express putative Ixodes scapularis antifreeze glycoprotein (IAFGP) and show that the presence of IAFGP increases the ability of flies to survive in the cold. Male and female adult iafgp-expressing D. melanogaster exhibited higher survival rates compared with controls when placed at non-freezing temperatures. Increased hatching rates were evident in embryos expressing IAFGP when exposed to the cold. The TUNEL assay showed that flight muscles from iafgp-expressing female adult flies exhibited less apoptotic damage upon exposure to non-freezing temperatures in comparison to control flies. Collectively, these data suggest that expression of iafgp increases cold tolerance in flies by preventing apoptosis. This study defines a molecular basis for the role of an antifreeze protein in cryoprotection of flies. PMID:22428051

  5. Antifreeze effect of carboxylated ε-poly-L-lysine on the growth kinetics of ice crystals.

    PubMed

    Vorontsov, Dmitry A; Sazaki, Gen; Hyon, Suong-Hyu; Matsumura, Kazuaki; Furukawa, Yoshinori

    2014-08-28

    Some biological substances control the nucleation and growth of inorganic crystals. Antifreeze proteins, which prohibit ice crystal growth in living organisms, promise are also important as biological antifreezes for medical applications and in the frozen food industries. In this work, we investigated the crystallization of ice in the presence of a new cryoprotector, carboxylated ε-poly-L-lysine (COOH-PLL). In order to reveal the characteristics and the mechanism of its antifreeze effect, free-growth experiments of ice crystals were carried out in solutions with various COOH-PLL concentrations and degrees of supercooling, and the depression of the freezing point and growth rates of the tips of ice dendrites were obtained using optical microscopy. Hysteresis of growth rates and depression of the freezing point was revealed in the presence of COOH-PLL. The growth-inhibition effect of COOH-PLL molecules could be explained on the basis of the Gibbs-Thomson law and the use of Langmuir's adsorption isotherm. Theoretical kinetic curves for hysteresis calculated on the basis of Punin-Artamonova's model were in good agreement with experimental data. We conclude that adsorption of large biological molecules in the case of ice crystallization has a non-steady-state character and occurs more slowly than the process of embedding of crystal growth units.

  6. Conformational and dynamic properties of a 14 residue antifreeze glycopeptide from Antarctic cod.

    PubMed Central

    Lane, A. N.; Hays, L. M.; Feeney, R. E.; Crowe, L. M.; Crowe, J. H.

    1998-01-01

    The 1H and 13C NMR spectra of a 14-residue antifreeze glycopeptide from Antarctic cod (Tetramatomnus borchgrevinki) containing two proline residues have been assigned. 13C NMR relaxation experiments indicate motional anisotropy of the peptide, with a tumbling time in water at 5 degrees C of 3-4 ns. The relaxation data and lack of long-range NOEs are consistent with a linear peptide undergoing significant segmental motion. However, extreme values of some coupling constants and strong sequential NOEs indicate regions of local order, which are most evident at the two ATPA subsequences. Similar spectroscopic properties were observed in the 16-residue analogue containing an Arg-Ala dipeptide added to the C-terminus. Molecular modeling also showed no evidence of long-range order, but the two ATPA subsequences were relatively well determined by the experimental data. These motifs were quite distinct from helical structures or beta turns commonly found in proteins, but rather resemble sections of an extended polyproline helix. Thus, the NMR data provide a description of the local order, which is of relevance to the mechanism of action of the antifreeze activity of the antifreeze glycopeptides as well as their ability to protect cells during hypothermic storage. PMID:9684888

  7. Conformational and dynamic properties of a 14 residue antifreeze glycopeptide from Antarctic cod.

    PubMed

    Lane, A N; Hays, L M; Feeney, R E; Crowe, L M; Crowe, J H

    1998-07-01

    The 1H and 13C NMR spectra of a 14-residue antifreeze glycopeptide from Antarctic cod (Tetramatomnus borchgrevinki) containing two proline residues have been assigned. 13C NMR relaxation experiments indicate motional anisotropy of the peptide, with a tumbling time in water at 5 degrees C of 3-4 ns. The relaxation data and lack of long-range NOEs are consistent with a linear peptide undergoing significant segmental motion. However, extreme values of some coupling constants and strong sequential NOEs indicate regions of local order, which are most evident at the two ATPA subsequences. Similar spectroscopic properties were observed in the 16-residue analogue containing an Arg-Ala dipeptide added to the C-terminus. Molecular modeling also showed no evidence of long-range order, but the two ATPA subsequences were relatively well determined by the experimental data. These motifs were quite distinct from helical structures or beta turns commonly found in proteins, but rather resemble sections of an extended polyproline helix. Thus, the NMR data provide a description of the local order, which is of relevance to the mechanism of action of the antifreeze activity of the antifreeze glycopeptides as well as their ability to protect cells during hypothermic storage.

  8. Lectin-like ox-LDL receptor-1 (LOX-1)-Toll-like receptor 4 (TLR4) interaction and autophagy in CATH.a differentiated cells exposed to angiotensin II.

    PubMed

    Ding, Zufeng; Liu, Shijie; Wang, Xianwei; Khaidakov, Magomed; Dai, Yao; Deng, Xiaoyan; Fan, Yubo; Xiang, David; Mehta, Jawahar L

    2015-04-01

    Toll-like receptors (TLRs) play an essential role in innate immune response. Expression of TLRs has also been linked to autophagy. As the main receptor for oxidized low-density lipoprotein (ox-LDL) on the cell surface, lectin-like ox-LDL receptor-1 (LOX-1) is upregulated by proinflammatory cytokines and has been linked to the development of autophagy. However, the relationship between LOX-1, autophagy, and TLR4 in neurons has not been defined. Here, we show that Angiotensin II (Ang II) treatment of CATH.a differentiated neuronal cells resulted in the expression of TLR4 (and associated signals MyD88 and Toll/interleukin-1 receptor domain-containing adapter-inducing interferon (TRIF)), LOX-1 autophagy. LOX-1 knockdown (transfection with specific small interfering RNA (siRNA)) resulted in reduced expression of TLR4 (and associated signals MyD88 and TRIF) and P-P38 mitogen-activated protein kinase (MAPK) and autophagy. TLR4 knockdown with siRNA resulted in reduced LOX-1 expression and autophagy, indicating a positive feedback between LOX-1 and TLR4. Knockdown of TRIF as well as MyD88 or inhibition of P38 MAPK also inhibited the expression of LOX-1 and TLR4 and autophagy. Importantly, pretreatment with 3-methyladenine (autophagy inhibitor) enhanced while rapamycin (autophagy inducer) decreased the expression of LOX-1, TLR4, and P-P38 MAPK. These studies suggest the presence of a bidirectional link between LOX-1and TLR4 in cultured CATH.a differentiated cells exposed to Ang II with an important role for autophagy in this link.

  9. Ethanol extract of propolis protects endothelial cells from oxidized low density lipoprotein-induced injury by inhibiting lectin-like oxidized low density lipoprotein receptor-1-mediated oxidative stress.

    PubMed

    Fang, Yongqi; Li, Jinguo; Ding, Mingde; Xu, Xiaoyan; Zhang, Jiajun; Jiao, Peng; Han, Ping; Wang, Jiafu; Yao, Shutong

    2014-12-01

    Lectin-like oxidized low density lipoprotein receptor-1 (LOX-1), as the primary oxidized low-density lipoprotein (ox-LDL) receptor on endothelial cells, plays a crucial role in endothelial injury, which is a driving force in the initiation and development of atherosclerosis. Our previous studies have shown that ethanol extract of propolis (EEP) promotes reverse cholesterol transport and inhibits atherosclerotic lesion development. However, the protective effects of EEP against ox-LDL-induced injury in endothelial cells and the underlying mechanisms are still unknown. This study was designed to test the hypothesis that EEP attenuates ox-LDL-induced endothelial oxidative injury via modulation of LOX-1-mediated oxidative stress. Our results showed that exposure of human umbilical vein endothelial cells (HUVECs) to ox-LDL (100 mg/L) led to the decrease in cell viability and increase in lactate dehydrogenase (LDH) release, caspase-3 activation, and apoptosis, whereas pretreatment with EEP (7.5, 15 and 30 mg/L) protected against such damages in a dose-dependent manner. In addition, EEP mitigated ox-LDL uptake by HUVECs and attenuated ox-LDL-upregulated LOX-1 expression both at the mRNA and protein levels. Moreover, EEP suppressed the ox-LDL-induced oxidative stress as assessed by decreased nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation, reactive oxygen species (ROS), and malondialdehyde (MDA) generation as well as increased antioxidant enzyme activities. Similar results were observed in the anti-LOX-1 antibody or diphenyleneiodonium (DPI)-pretreated HUVECs. These data indicate that EEP may protect HUVECs from ox-LDL-induced injury and that the mechanism at least partially involves its ability to inhibit endothelial LOX-1 upregulation and subsequent oxidative stress.

  10. Inhibition of bacterial ice nucleators by fish antifreeze glycoproteins.

    PubMed

    Parody-Morreale, A; Murphy, K P; Di Cera, E; Fall, R; DeVries, A L; Gill, S J

    1988-06-23

    Certain bacteria promote the formation of ice in super-cooled water by means of ice nucleators which contain a unique protein associated with the cell membrane. Ice nucleators in general are believed to act by mimicking the structure of an ice crystal surface, thus imposing an ice-like arrangement on the water molecules in contact with the nucleating surface and lowering the energy necessary for the initiation of ice formation. Quantitative investigation of the bacterial ice-nucleating process has recently been made possible by the discovery of certain bacteria that shed stable membrane vesicles with ice nucleating activity. The opposite effect, inhibition of ice formation, has been described for a group of glycoproteins found in different fish and insect species. This group of substances, termed antifreeze glycoproteins (AFGPs), promotes the supercooling of water with no appreciable effect on the equilibrium freezing point or melting temperature. Substantial evidence now indicates that AFGPs act by binding to a growing ice crystal and slowing crystal growth. As the ice-nucleating protein surface is believed to have a structure similar to an embryonic ice crystal, AFGPs might be predicted to interact directly with a bacterial ice-nucleating site. We report here that AFGPs from the antarctic fish Dissostichus mawsoni inhibit the ice-nucleating activity of membrane vesicles from the bacterium Erwinia herbicola. The inhibition effect shows saturation at high concentration of AFGP and conforms to a simple binding reaction between the AFGP and the nucleation centre.

  11. Restraint stress up-regulates lectin-like oxidized low-density lipoprotein receptor-1 in aorta of apolipoprotein E-deficient mice.

    PubMed

    Andersson, Irene J; Sankaralingam, Sowndramalingam; Davidge, Sandra T

    2010-09-01

    Psychological stress is a risk factor for cardiovascular disease including atherosclerosis, but the mechanisms are unknown. The vascular lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is involved in vascular pathology and early atherogenesis. We hypothesized that LOX-1 is up-regulated by psychological stress via the formation of oxygen-derived free radicals, and that treatment with EUK-8 (a superoxide dismutase and catalase mimetic) prevents production of oxygen-derived free radicals and leads to reduced expression of LOX-1 in the vascular wall. As a model for psychological stress, we exposed male apolipoprotein E-deficient mice to repeated restraint stress by placement in a conical tube for 2 h per day for 14 consecutive days. Stressed and control mice were treated with EUK-8 (n = 4-5) or vehicle (n = 4-5). Reactive oxygen species and peroxynitrite levels, as detected by oxidative fluorescence microscopy, were increased in the aortic root of mice exposed to stress compared to those of controls by 212 +/- 22% (mean +/- SEM; p < 0.001) and 110 +/- 6% (p < 0.001), respectively. LOX-1, as detected by immunohistochemistry, was increased by 443 +/- 63% in stressed mice compared to control mice (p < 0.001). EUK-8 reduced reactive oxygen species, peroxynitrite, and LOX-1 levels in stressed mice compared to vehicle-treated stressed mice. To conclude, LOX-1 induced by reactive oxygen species and/or peroxynitrite could be one mechanism by which stress promotes cardiovascular disease.

  12. Lectin-like Oxidized Low-Density Lipoprotein (LDL) Receptor (LOX-1): A Chameleon Receptor for Oxidized LDL.

    PubMed

    Zeya, Bushra; Arjuman, Albina; Chandra, Nimai Chand

    2016-08-16

    LOX-1, one of the main receptors for oxLDL, is found mainly on the surface of endothelial cells. It is a multifacet 52 kDa type II transmembrane protein that structurally belongs to the C-type lectin family. It exists with short intracellular N-terminal and long extracellular C-terminal hydrophilic domains separated by a hydrophobic domain of 26 amino acids. LOX-1 acts like a bifunctional receptor either showing pro-atherogenicity by activating the NFκB-mediated down signaling cascade for gene activation of pro-inflammatory molecules or playing an atheroprotective agent by receptor-mediated uptake of oxLDL in the presence of an anti-inflammatory molecule like IL-10. Mildly, moderately, and highly oxidized LDL show their characteristic features upon LOX-1 activation and its ligand binding indenture. The polymorphic LOX-1 genes are intensively associated with increased susceptibility to myocardial diseases. The splicing variant LOX IN dimerizes with the native form of LOX-1 and protects cells from damage by oxidized LDL. In the developing field of regenerating medicine, LOX-1 is a potential target for therapeutic intervention.

  13. The N-terminal of a heparin-binding sperm membrane mitogen possess lectin-like sequence

    SciTech Connect

    Mor, Visesato; Chatterjee, Tapati . E-mail: c_tapati@yahoo.com

    2007-03-02

    Glycosaminoglycans like heparin and heparin sulfate in follicular fluid induce changes in the intracellular environment during the spermatozoal functional maturation. We previously reported the isolation, purification and partial characterization of a heparin binding sperm membrane protein (HBSM). In the present study, the amino acids analysis provided evidence of a single sequence, which suggest the homogeneity of the purified HBSM. Fourteen amino acids-{sup 1} A D T I V A V E L D T Y P N {sup 14}-correspond to the amino terminal sequence of Concanavalin A (Con A) and contain 45.2% carbohydrate by weight. HBSM possess mitogenic property on lymphocytes with comparable magnitude to the well-known mitogen; Con A, inducing 83% radiolabel thymidine incorporation in growing lymphocytes. Unlike Con A, there was no agglutination of cell by HBSM upto 5 ng/ml concentration. Interestingly, we found that heparin and chondroitin sulfate-conjugated HBSM inhibit the proliferative activity. Similar effect was also found with an in-house isolate sulfated glycans; G-I (28% sulfate). In contrast, there was no inhibition by the desulfated form; G-ID. Altogether, our data suggest that the mechanism of cell proliferative pathway may be different for HBSM and Con A.

  14. A novel gene silencer, pyrrole-imidazole polyamide targeting human lectin-like oxidized low-density lipoprotein receptor-1 gene improves endothelial cell function.

    PubMed

    Ueno, Takahiro; Fukuda, Noboru; Tsunemi, Akiko; Yao, En-Hui; Matsuda, Hiroyuki; Tahira, Kazunobu; Matsumoto, Taro; Matsumoto, Koichi; Matsumoto, Yoshiaki; Nagase, Hiroki; Sugiyama, Hiroshi; Sawamura, Tatsuya

    2009-03-01

    Pyrrole-imidazole polyamide can be combined in antiparallel side-by-side dimeric complexes along the minor groove of DNA in a sequence-specific manner. Pyrrole-imidazole polyamides are effective inhibitors of transcription factors as well as viral repressors and transactivators. Recently, lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) was reported to be a major factor contributing to the pathogenesis of coronary atherosclerosis. In this study, we designed a pyrrole-imidazole polyamide specific for the LOX-1 gene and evaluated its effect on LOX-1 gene transcription. A pyrrole-imidazole polyamide was designed to target the AP-1 binding site of the LOX-1 gene and synthesized by solid phase methods. This pyrrole-imidazole polyamide significantly inhibited LOX-1 promoter activity in HEK293 cells, determined by the luciferase assay. LOX-1 mRNA expression was also inhibited by the pyrrole-imidazole polyamide at a concentration of 10-9 mol/l in human umbilical vein endothelial cells (HUVEC), determined by the real-time PCR method. HUVEC were treated by pyrrole-imidazole polyamide targeting the LOX-1 gene, and apoptosis was assessed using Hoechst stain, terminal deoxy nucleotidyl transferase-mediated UTP end labeling method, and dye-uptake bioassay. Treatment of HUVEC for 72 h with LOX-1 targeted pyrrole-imidazole polyamide decreased apoptosis induced by angiotensin II and oxidized low-density lipoprotein (ox-LDL) loading in all assays. This novel therapeutic agent, pyrrole-imidazole polyamide, could specifically inhibit LOX-1 gene expression by reducing the promoter activity of the gene. Pyrrole-imidazole polyamide seems to be a powerful promising new agent that can be used to explore therapies based on inhibition of transcription. Molecular recognition of DNA by small molecules could provide insight into the development of new human medicines.

  15. The CD94/NKG2C killer lectin-like receptor constitutes an alternative activation pathway for a subset of CD8+ T cells.

    PubMed

    Gumá, Mónica; Busch, Lisa K; Salazar-Fontana, Laura I; Bellosillo, Beatriz; Morte, Carles; García, Pilar; López-Botet, Miguel

    2005-07-01

    The CD94/NKG2C killer lectin-like receptor (KLR) specific for HLA-E is coupled to the KARAP/DAP12 adapter in a subset of NK cells, triggering their effector functions. We have studied the distribution and function of this KLR in T lymphocytes. Like other NK cell receptors (NKR), CD94/NKG2C was predominantly expressed by a CD8(+) T cell subset, though TCRgammadelta(+) NKG2C(+) and rare CD4(+) NKG2C(+) cells were also detected in some individuals. Coculture with the 721.221 HLA class I-deficient lymphoma cell line transfected with HLA-E (.221-AEH) induced IL-2Ralpha expression in CD94/NKG2C+ NK cells and a minor subset of CD94/NKG2C(+) T cells, promoting their proliferation; moreover, a similar response was triggered upon selective engagement of CD94/NKG2C with a specific mAb. CD8(+) TCRalphabeta CD94/NKG2C(+) T cell clones, that displayed different combinations of KIR and CD85j receptors, expressed KARAP/DAP12 which was co-precipitated by an anti-CD94 mAb. Specific engagement of the KLR triggered cytotoxicity and cytokine production in CD94/NKG2C(+) T cell clones, inducing as well IL-2Ralpha expression and a proliferative response. Altogether these results support that CD94/NKG2C may constitute an alternative T cell activation pathway capable of driving the expansion and triggering the effector functions of a CTL subset.

  16. High Levels of Soluble Lectin-Like Oxidized Low-Density Lipoprotein Receptor-1 in Acute Stroke: An Age- and Sex-Matched Cross-Sectional Study

    PubMed Central

    Sawamura, Tatsuya; Watanabe, Makoto; Kokubo, Yoshihiro; Fujita, Yoshiko; Kakino, Akemi; Nakai, Michikazu; Toyoda, Kazunori; Miyamoto, Yoshihiro; Minematsu, Kazuo

    2016-01-01

    Aim: Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is known to be a key molecule in the pathogenesis of atherosclerosis. Although high levels of serum soluble LOX-1 (sLOX-1) were demonstrated in patients with acute coronary syndrome, there are no reports about acute stroke patients. The aim of the present study was to evaluate the levels of sLOX-1 in acute stroke patients according to different stroke subtypes. Methods: We enrolled a total of 377 patients with a stroke (men/women: 251/126; age: 40–79 years), 250 with ischemic stroke and 127 with intracerebral hemorrhage (ICH). Patients were admitted to our hospital within 3 days after the onset of stroke. As controls, we randomly selected age- and sex-matched subjects without a past history of cardiovascular disease according to stroke subtype from the community-based cohort of the Suita study. Serum LOX-1 levels were compared between stroke patients and healthy controls according to stroke subtype. Results: Median values of serum sLOX-1 in stroke patients were significantly higher than those in controls (526 vs. 486 ng/L in ischemic stroke and 720 vs. 513 ng/L in ICH, respectively). Among subtypes of ischemic stroke, median sLOX-1 levels in atherothrombotic brain infarction (641 ng/L) only were significantly higher than those in controls (496 ng/L). Ischemic stroke [odds ratio (OR), 3.80; 95% confidence interval (CI), 1.86–7.74] and ICH (OR, 5.97; 95% CI, 2.13–16.77) were independently associated with high levels of sLOX-1 by multivariate logistic regression analysis. Conclusions: Higher levels of sLOX-1 were observed in patients with acute stoke than in controls. High levels of sLOX-1 can be useful as biomarker for acute stroke. PMID:27025681

  17. Bioinspired Antifreeze Secreting Frost-Responsive Pagophobic Coatings

    NASA Astrophysics Data System (ADS)

    Sun, Xiaoda; Damle, Viraj; Rykaczewski, Konrad

    2014-11-01

    Prevention of ice and frost accumulation is of interest to transportation, power generation, and agriculture industries. Superhydrophobic and lubricant impregnated pagophobic coatings have been proposed, however, they both fail in frosting conditions. Inspired by functional liquid secretion in natural systems, such as toxin secretion by poison dart frost in response to predator presence, we developed frost-responsive antifreeze secreting pagophobic coatings. These are bi-layered coatings with an inner superhydrophilic ``dermis'' infused with antifreeze and an outer permeable superhydrophobic ``epidermis.'' The superhydrophobic epidermis separates the antifreeze from the environment and prevents ice accumulation by repelling impinging water droplets. In frosting conditions, the antifreeze is secreted from the dermis through pores in the epidermis either due to contact with condensed droplets or temporary switch of the epidermis wettability from hydrophobic to hydrophilic caused by surface icing. Here we demonstrate superior performance of this multifunctional coating in simulated frosting, freezing mist/fog, and freezing spray/rain conditions. KR acknowledges startup funding from ASU.

  18. The cryoprotective effect of antifreeze glycopeptides from antarctic fishes.

    PubMed

    Rubinsky, B; Arav, A; Devries, A L

    1992-02-01

    Apparently vitrified cells and tissues often fail to survive, probably from damage from growth of microscopically invisible ice crystals. Special biological antifreezes from some polar fishes have been shown to adsorb to specific faces of ice crystals and inhibit crystal growth. Vitrification in the presence of antifreezes therefore may help enhance postvitrification viability of cells and tissues. We report here that the addition of fish antifreeze glycopeptides (AFGPs) to vitrifying solutions increases post-thaw viability in cultured immature pig oocytes and two-cell stage embryos of mice and pigs after rapid cooling to cryogenic temperatures. The criterion for viability is maturation to metaphase for the oocytes and the ability to develop into the four-cell stage for the pig embryo and the blastocyst stage for the mouse embryo. Without AFGPs, or with addition of antifreeze peptides (AFPs), the particular vitrifying solution and cooling/warming/culturing regime used in this study produced zero viability. In the presence of the AFGPs (40 mg/ml), survival of pig oocytes and embryos was increased to about 25%, and that of mouse embryos to 82%. Dose-response studies for the mouse embryos showed that the protective effect of AFGPs shows saturation kinetics and levels off at 20 mg/ml. The AFGPs appeared to preserve cell membrane structural integrity; however, an intact cell membrane did not always lead to viability. The absence of protective effect by AFPs suggests that protection by the AFGPs is unrelated to their common antifreeze property, i.e., inhibition of ice crystal growth, but probably results from interaction with and stabilization of the cell membranes unique to the AFGPs.

  19. Antifreeze and cryoprotective activities of ice-binding collagen peptides from pig skin.

    PubMed

    Cao, Hui; Zhao, Ying; Zhu, Yu Bing; Xu, Fei; Yu, Jing Song; Yuan, Min

    2016-03-01

    A novel "hyperactive" ice-binding peptide from porcine collagen was prepared by alkaline protease hydrolysis and a series of column chromatography separations, and then its antifreeze and cryoprotective properties were reported. Using differential scanning calorimetry (DSC), the thermal hysteresis (TH) of ice-binding collagen peptides was closely related to their concentration and crystal fraction. Collagen hydrolysates with maximal TH were obtained by hydrolysis at pH 8.0, DH 15.0%, and 5% alkaline protease at 55°C. After purification by column chromatography, the AP-3 ice-binding collagen peptide (GLLGPLGPRGLL) with 1162.8Da molecular weights exhibited the highest TH (5.28°C), which can be classified as "hyperactive". Recrystallisation and melt-resistance of ice cream were improved by AP-3 ice-binding collagen peptide at 0.2% (w/v) in a similar manner to natural antifreeze proteins. Moreover, the addition of AP-3 collagen peptides in ice cream greatly elevated the glass transition temperature (Tg) to -17.64°C.

  20. Increased serum soluble lectin-like oxidized low-density lipoprotein receptor-1 levels in patients with biopsy-proven nonalcoholic fatty liver disease

    PubMed Central

    Ozturk, Oguzhan; Colak, Yasar; Senates, Ebubekir; Yilmaz, Yusuf; Ulasoglu, Celal; Doganay, Levent; Ozkanli, Seyma; Oltulu, Yasemin Musteri; Coskunpinar, Ender; Tuncer, Ilyas

    2015-01-01

    AIM: To analyze the relationship between the serum lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) levels and clinical and histopathological features of biopsy-confirmed nonalcoholic fatty liver disease (NAFLD) patients. METHODS: Fifty-three consecutive, biopsy-proven NAFLD patients (31 males and 22 females, mean age 42.5 ± 9.6 years) and 26 age- and gender-matched, healthy controls (14 males and 12 females, mean age 39 ± 10.7 years) were included. The patients with NAFLD were consecutive patients who had been admitted to the hepatology outpatient clinic within the last year and had been diagnosed with NAFLD as the result of liver biopsy. The healthy controls were individuals who attended the outpatient clinic for routine health control and had no known chronic illnesses. The histological evaluation was conducted according to the NAFLD activity scoring system recommended by The National Institute of Diabetes and Digestive and Kidney Diseases Nonalcoholic Steatohepatitis Clinical Research Network. The serum LOX-1 levels were measured using an ELISA kit (Life Science Inc. USCN. Wuhan, Catalog No. E1859Hu) in both patients and healthy controls. A receiver operating characteristic (ROC) curve analysis was used to identify the optimal cutoff value of LOX-1 and thereby distinguish between patients with nonalcoholic steatohepatitis (NASH) and healthy controls. A P-value < 0.05 was considered statistically significant. RESULTS: NAFLD and healthy control groups were similar in terms of age and sex. NAFLD patients consisted of 8 patients with simple steatosis (15%), 27 with borderline NASH (51%) and 18 with definitive NASH (34%). Metabolic syndrome was found in 62.2% of the patients with NAFLD. The mean serum LOX-1 level in biopsy-proven NAFLD patients was 8.49 ± 6.43 ng/mL compared to 4.08 ± 4.32 ng/mL in healthy controls (P = 0.001). The LOX-1 levels were significantly different between controls, simple steatosis and NASH (borderline+definite) cases (4

  1. Variations in macromolecular antifreeze levels in larvae of the darkling beetle, Meracantha contracta.

    PubMed

    Duman, J G

    1977-07-01

    Overwintering larvae of the darkling beetle, Meracantha contracta, produce a macromolecular antifreeze that is similar in activity to the glycoproteinaceous and proteinaceous antifreezes found in some cold-water, marine teleost fishes. The antifreeze is not present in the hemolymph of the Meracantha larvae in summer, but its production begins by late September in the wild population. The antifreeze reaches a maximum concentration in February, decreases slowly through spring, and disappears by early June. The supercooling points of the larvae are lowest in February, when the antifreeze levels are highest, and increase as the antifreeze concentrations in the hemolymph decrease in the spring. Larvae collected in mid-February and warm-acclimated lost the antifreeze with-in 12 days. Larvae collected in early September and cold-acclimated required nearly two months to produce concentrations of antifreeze comparable to those of overwintering larvae. Temperature seems to be the major environmental factor responsible for the control of antifreeze levels in Meracantha; however, other environmental factors may also be involved.

  2. Ice growth in supercooled solutions of antifreeze glycoproteins

    NASA Technical Reports Server (NTRS)

    Harrison, K.; Hallett, J.; Burcham, T. S.; Feeney, R. E.; Kerr, W. L.

    1987-01-01

    The effects of different degrees of supercooling on the habit and rates of growth of ice crystals from solutions of antifreeze glycoproteins are reported. To isolate the influence of different solutions and supercooling alone, a system was devised that nucleated crystals in the middle of a uniformly supercooled sample. Alternatively, single crystals of selected orientation were inserted into free liquid surface. A crystallization rate up to five times greater than that in pure water was found. A mechanism explaining these results is suggested.

  3. Isolation of an antifreeze peptide from the Antarctic sponge Homaxinella balfourensis

    PubMed Central

    Wilkins, S. P.; Blum, A. J.; Burkepile, D. E.; Rutland, T. J.; Wierzbicki, A.; Kelly, M.; Hamann, M.T.

    2016-01-01

    Polar plants and animals survive in subzero waters (−2°C) and many of these marine organisms produce antifreeze proteins (AFPs) to better adapt themselves to these conditions. AFPs prevent the growth of ice crystals which disrupt cellular membranes and destroy cells by inhibiting crystallization of water within the organism. The hydrophilic extract of an Antarctic sponge Homaxinella balfourensis exhibited a non-colligative freezing point depression effect on the crystal morphology of water. The extract was purified by repeated reverse phase high-pressure liquid chromatography, then assayed and shown to contain several AFPs. The major peptide was isolated, analyzed using matrix-assisted laser desorption ionization mass spectrometry and the partial structure of the peptide identified through amino acid sequencing. AFPs have potential applications in agriculture, medicine and the food industry. PMID:12568347

  4. Integration of Nanofluids into Commercial Antifreeze Concentrates with ASTM D15 Corrosion Testing

    DTIC Science & Technology

    2013-05-01

    Nanofluids in Ethylene Glycol /Water Solution The first stage evaluation was focused on dispersion stability and thermal conductivity increase (which...practical for use. The first stage evaluation was not tried with commercial antifreeze concentrate (Zerex Antifreeze). Instead pure ethylene glycol ...Lockwood1 1 Valvoline New Product Development Laboratory, Ashland Consumer Markets, Lexington, Kentucky. 2 Tank Automotive Research, Development

  5. Bacterial ice crystal controlling proteins.

    PubMed

    Lorv, Janet S H; Rose, David R; Glick, Bernard R

    2014-01-01

    Across the world, many ice active bacteria utilize ice crystal controlling proteins for aid in freezing tolerance at subzero temperatures. Ice crystal controlling proteins include both antifreeze and ice nucleation proteins. Antifreeze proteins minimize freezing damage by inhibiting growth of large ice crystals, while ice nucleation proteins induce formation of embryonic ice crystals. Although both protein classes have differing functions, these proteins use the same ice binding mechanisms. Rather than direct binding, it is probable that these protein classes create an ice surface prior to ice crystal surface adsorption. Function is differentiated by molecular size of the protein. This paper reviews the similar and different aspects of bacterial antifreeze and ice nucleation proteins, the role of these proteins in freezing tolerance, prevalence of these proteins in psychrophiles, and current mechanisms of protein-ice interactions.

  6. Bacterial Ice Crystal Controlling Proteins

    PubMed Central

    Lorv, Janet S. H.; Rose, David R.; Glick, Bernard R.

    2014-01-01

    Across the world, many ice active bacteria utilize ice crystal controlling proteins for aid in freezing tolerance at subzero temperatures. Ice crystal controlling proteins include both antifreeze and ice nucleation proteins. Antifreeze proteins minimize freezing damage by inhibiting growth of large ice crystals, while ice nucleation proteins induce formation of embryonic ice crystals. Although both protein classes have differing functions, these proteins use the same ice binding mechanisms. Rather than direct binding, it is probable that these protein classes create an ice surface prior to ice crystal surface adsorption. Function is differentiated by molecular size of the protein. This paper reviews the similar and different aspects of bacterial antifreeze and ice nucleation proteins, the role of these proteins in freezing tolerance, prevalence of these proteins in psychrophiles, and current mechanisms of protein-ice interactions. PMID:24579057

  7. Oscillations and accelerations of ice crystal growth rates in microgravity in presence of antifreeze glycoprotein impurity in supercooled water.

    PubMed

    Furukawa, Yoshinori; Nagashima, Ken; Nakatsubo, Shun-Ichi; Yoshizaki, Izumi; Tamaru, Haruka; Shimaoka, Taro; Sone, Takehiko; Yokoyama, Etsuro; Zepeda, Salvador; Terasawa, Takanori; Asakawa, Harutoshi; Murata, Ken-Ichiro; Sazaki, Gen

    2017-03-06

    The free growth of ice crystals in supercooled bulk water containing an impurity of glycoprotein, a bio-macromolecule that functions as 'antifreeze' in living organisms in a subzero environment, was observed under microgravity conditions on the International Space Station. We observed the acceleration and oscillation of the normal growth rates as a result of the interfacial adsorption of these protein molecules, which is a newly discovered impurity effect for crystal growth. As the convection caused by gravity may mitigate or modify this effect, secure observations of this effect were first made possible by continuous measurements of normal growth rates under long-term microgravity condition realized only in the spacecraft. Our findings will lead to a better understanding of a novel kinetic process for growth oscillation in relation to growth promotion due to the adsorption of protein molecules and will shed light on the role that crystal growth kinetics has in the onset of the mysterious antifreeze effect in living organisms, namely, how this protein may prevent fish freezing.

  8. Antifreeze Activity of Xylomannan from the Mycelium and Fruit Body of Flammulina velutipes.

    PubMed

    Kawahara, Hidehisa; Matsuda, Yoshiyuki; Sakaguchi, Takuya; Arai, Naoki; Koide, Yoshihide

    2016-01-01

    An identified class of antifreeze, a xylomannan-based thermal hysteresis (TH)-producing glycolipid, has been discovered from diverse taxa, including plants, insects, and amphibians. We isolated xylomannan from the mycelium and fruit body of the basidiomycete Flammulina velutipes using successive hot extraction with water, 2% and 25% aqueous KOH, and gel filtration chromatography. The xylomannan from the fruit body had a recrystallization inhibiting (RI) activity (RI=0.44) at 0.5 mg/mL. The dried weight yield of the fruit body (7.7×10(-2)%, w/w) was higher than that of the mycelium. Although the purified xylomannan from both soures were composed of mannose and xylose in a 2 : 1 molar ratio, the molecular weight of the xylomannan from the mycelium and fruit body was 320,000 and 240,000, respectively. The RI activity of mycelial xylomannan was higher than that from the fruit body (RI=0.57) at 45 µg/mL. Although this RI activity was able to remain constant after exposure to various conditions, we confirmed that the decrease of RI activity was stimulated by the decrease of molecular weight that was caused by heating during the alkaline condition. The survival rate of the CHO cells at -20℃ for two days increased to 97% due to the addition of 20 µg/mL of purified xylomannan. This was the first report to indicate that xylomannan from the mycelium of Flammulina velutipes had a high level of ice recrystallization inhibiting activity like antifreeze proteins from plants and had rhe potential to become a new material for cell storage.

  9. Solution Structure and Sugar-Binding Mechanism of Mouse Latrophilin-1 RBL: a 7TM Receptor-Attached Lectin-Like Domain

    PubMed Central

    Vakonakis, Ioannis; Langenhan, Tobias; Prömel, Simone; Russ, Andreas; Campbell, Iain D.

    2008-01-01

    Summary Latrophilin-1 (Lat-1), a target receptor for α-Latrotoxin, is a putative G protein-coupled receptor implicated in synaptic function. The extracellular portion of Lat-1 contains a rhamnose binding lectin (RBL)-like domain of unknown structure. RBL domains, first isolated from the eggs of marine species, are also found in the ectodomains of other metazoan transmembrane proteins, including a recently discovered coreceptor of the neuronal axon guidance molecule SLT-1/Slit. Here, we describe a structure of this domain from the mouse Lat-1. RBL adopts a unique α/β fold with long structured loops important for monosaccharide recognition, as shown in the structure of a complex with L-rhamnose. Sequence alignments and mutagenesis show that residues important for carbohydrate binding are often absent in other receptor-attached examples of RBL, including the SLT-1/Slit coreceptor. We postulate that this domain class facilitates direct protein-protein interactions in many transmembrane receptors. PMID:18547526

  10. Lectin-like domain of thrombomodulin binds to its specific ligand Lewis Y antigen and neutralizes lipopolysaccharide-induced inflammatory response

    PubMed Central

    Shi, Chung-Sheng; Hsiao, Shi-Ming; Kao, Yuan-Chung; Kuo, Kuan-Lin; Ma, Chih-Yuan; Kuo, Cheng-Hsiang; Chang, Bi-Ing; Chang, Chuan-Fa; Lin, Chun-Hung; Wong, Chi-Huey

    2008-01-01

    Thrombomodulin (TM), a widely expressing glycoprotein originally identified in vascular endothelium, is an important cofactor in the protein C anticoagulant system. TM appears to exhibit anti-inflammatory ability through both protein C–dependent and –independent pathways. We presently have demonstrated that recombinant N-terminal lectinlike domain of TM (rTMD1) functions as a protective agent against sepsis caused by Gram-negative bacterial infections. rTMD1 caused agglutination of Escherichia coli and Klebsiella pneumoniae and enhanced the macrophage phagocytosis of these Gram-negative bacteria. Moreover, rTMD1 bound to the Klebsiella pneumoniae and lipopolysaccharide (LPS) by specifically interacting with Lewis Y antigen. rTMD1 inhibited LPS-induced inflammatory mediator production via interference with CD14 and LPS binding. Furthermore, rTMD1 modulated LPS-induced mitogen-activated protein kinase and nuclear factor-κB signaling pathway activations and inducible nitric oxide synthase expression in macrophages. Administration of rTMD1 protected the host by suppressing inflammatory responses induced by LPS and Gram-negative bacteria, and enhanced LPS and bacterial clearance in sepsis. Thus, rTMD1 can be used to defend against bacterial infection and inhibit LPS-induced inflammatory responses, suggesting that rTMD1 may be valuable in the treatment of severe inflammation in sepsis, especially in Gram-negative bacterial infections. PMID:18711002

  11. Dihydrotanshinone I Attenuates Atherosclerosis in ApoE-Deficient Mice: Role of NOX4/NF-κB Mediated Lectin-Like Oxidized LDL Receptor-1 (LOX-1) of the Endothelium.

    PubMed

    Zhao, Wenwen; Li, Chunxia; Gao, Hongwei; Wu, Qin; Shi, Jingshan; Chen, Xiuping

    2016-01-01

    Dihydrotanshinone I (DHT) is a natural compound extracted from Salvia miltiorrhiza Bunge which has been widely used for treating cardiovascular diseases. However, its role in atherosclerosis remains unclear. In this study, the effect of DHT on atherosclerosis were investigated using apolipoprotein E-deficient (ApoE(-/-)) mice and endothelial cells. In lipopolysaccharide (LPS)-stimulated human umbilical vein endothelial cells (HUVECs), DHT (10 nM) decreased lectin-like ox-LDL receptor-1 (LOX-1) and NADPH oxidase 4 (NOX4) expression, reactive oxygen species (ROS) production, NF-κB nuclear translocation, ox-LDL endocytosis and monocytes adhesion. Silence NOX4 inhibited LPS-induced LOX-1 expression, NF-κB nuclear translocation, ox-LDL endocytosis and monocytes adhesion. In ApoE(-/-) mice fed with an atherogenic diet, DHT (10 and 25 mg kg(-1)) significantly attenuated atherosclerotic plaque formation, altered serum lipid profile, decreased oxidative stress and shrunk necrotic core areas. The enhanced expression of LOX-1, NOX4, and NF-κB in aorta was also dramatically inhibited by DHT. In conclusion, these results suggested that DHT showed anti-atherosclerotic activity through inhibition of LOX-1 mediated by NOX4/NF-κB signaling pathways both in vitro and in vivo. This finding suggested that DHT might be used as a potential vascular protective candidate for the treatment of atherosclerosis.

  12. Dihydrotanshinone I Attenuates Atherosclerosis in ApoE-Deficient Mice: Role of NOX4/NF-κB Mediated Lectin-Like Oxidized LDL Receptor-1 (LOX-1) of the Endothelium

    PubMed Central

    Zhao, Wenwen; Li, Chunxia; Gao, Hongwei; Wu, Qin; Shi, Jingshan; Chen, Xiuping

    2016-01-01

    Dihydrotanshinone I (DHT) is a natural compound extracted from Salvia miltiorrhiza Bunge which has been widely used for treating cardiovascular diseases. However, its role in atherosclerosis remains unclear. In this study, the effect of DHT on atherosclerosis were investigated using apolipoprotein E-deficient (ApoE-/-) mice and endothelial cells. In lipopolysaccharide (LPS)-stimulated human umbilical vein endothelial cells (HUVECs), DHT (10 nM) decreased lectin-like ox-LDL receptor-1 (LOX-1) and NADPH oxidase 4 (NOX4) expression, reactive oxygen species (ROS) production, NF-κB nuclear translocation, ox-LDL endocytosis and monocytes adhesion. Silence NOX4 inhibited LPS-induced LOX-1 expression, NF-κB nuclear translocation, ox-LDL endocytosis and monocytes adhesion. In ApoE-/- mice fed with an atherogenic diet, DHT (10 and 25 mg kg-1) significantly attenuated atherosclerotic plaque formation, altered serum lipid profile, decreased oxidative stress and shrunk necrotic core areas. The enhanced expression of LOX-1, NOX4, and NF-κB in aorta was also dramatically inhibited by DHT. In conclusion, these results suggested that DHT showed anti-atherosclerotic activity through inhibition of LOX-1 mediated by NOX4/NF-κB signaling pathways both in vitro and in vivo. This finding suggested that DHT might be used as a potential vascular protective candidate for the treatment of atherosclerosis. PMID:27891092

  13. Attenuated natural killer (NK) cell activation through C-type lectin-like receptor NKp80 is due to an anomalous hemi-immunoreceptor tyrosine-based activation motif (HemITAM) with impaired Syk kinase recruitment capacity.

    PubMed

    Rückrich, Thomas; Steinle, Alexander

    2013-06-14

    Cellular cytotoxicity is the hallmark of NK cells mediating both elimination of virus-infected or malignant cells, and modulation of immune responses. NK cytotoxicity is triggered upon ligation of various activating NK cell receptors. Among these is the C-type lectin-like receptor NKp80 which is encoded in the human Natural Killer Gene Complex (NKC) adjacent to its ligand, activation-induced C-type lectin (AICL). NKp80-AICL interaction promotes cytolysis of malignant myeloid cells, but also stimulates the mutual crosstalk between NK cells and monocytes. While many activating NK cell receptors pair with ITAM-bearing adaptors, we recently reported that NKp80 signals via a hemITAM-like sequence in its cytoplasmic domain. Here we molecularly dissect the NKp80 hemITAM and demonstrate that two non-consensus amino acids, in particular arginine 6, critically impair both hemITAM phosphorylation and Syk recruitment. Impaired Syk recruitment results in a substantial attenuation of cytotoxic responses upon NKp80 ligation. Reconstituting the hemITAM consensus or Syk overexpression resulted in robust NKp80-mediated responsiveness. Collectively, our data provide a molecular rationale for the restrained activation potential of NKp80 and illustrate how subtle alterations in signaling motifs determine subsequent cellular responses. They also suggest that non-consensus alterations in the NKp80 hemITAM, as commonly present among mammalian NKp80 sequences, may have evolved to dampen NKp80-mediated cytotoxic responses toward AICL-expressing cells.

  14. Identification of a new pea gene, PsNlec1, encoding a lectin-like glycoprotein isolated from the symbiosomes of root nodules.

    PubMed Central

    Kardailsky, I V; Sherrier, D J; Brewin, N J

    1996-01-01

    A 27-kD glycoprotein antigen recognized by monoclonal antibody MAC266 was purified from isolated symbiosomes derived from pea (Pisum sativum) root nodules containing Rhizobium. The N-terminal amino acid sequence was obtained, and the corresponding cDNA clone was isolated by a polymerase chain reaction-based strategy. The clone contained a single open reading frame, and the gene was termed PsNlec1. Phylogenetic analysis of 31 legume sequences showed that the PsNlec1 protein is related to the legume lectin family but belongs to a subgroup that is very different from pea seed lectin. Expression of the PsNlec1 transcript was much stronger in nodules than in other parts of the plant. It was found in both infected and uninfected cells in the central tissue of the nodule and in the stele of the root near the attachment point of the nodule. When uninfected pea seedlings were grown on medium containing nitrate, weak transcription of PsNlec1 was observed in the root system. The identification of PsNlec1 inside the symbiosome is consistent with the observation that legume lectins are generally vacuolar proteins that may serve as transient storage components. PMID:8685275

  15. Inhibition of Condensation Frosting by Arrays of Hygroscopic Antifreeze Drops.

    PubMed

    Sun, Xiaoda; Damle, Viraj G; Uppal, Aastha; Linder, Rubin; Chandrashekar, Sriram; Mohan, Ajay R; Rykaczewski, Konrad

    2015-12-29

    The formation of frost and ice can have negative impacts on travel and a variety of industrial processes and is typically addressed by dispensing antifreeze substances such as salts and glycols. Despite the popularity of this anti-icing approach, some of the intricate underlying physical mechanisms are just being unraveled. For example, recent studies have shown that in addition to suppressing ice formation within its own volume, an individual salt saturated water microdroplet forms a region of inhibited condensation and condensation frosting (RIC) in its surrounding area. This occurs because salt saturated water, like most antifreeze substances, is hygroscopic and has water vapor pressure at its surface lower than water saturation pressure at the substrate. Here, we demonstrate that for macroscopic drops of propylene glycol and salt saturated water, the absolute RIC size can remain essentially unchanged for several hours. Utilizing this observation, we demonstrate that frost formation can be completely inhibited in-between microscopic and macroscopic arrays of propylene glycol and salt saturated water drops with spacing (S) smaller than twice the radius of the RIC (δ). Furthermore, by characterizing condensation frosting dynamics around various hygroscopic drop arrays, we demonstrate that they can delay complete frosting over of the samples 1.6 to 10 times longer than films of the liquids with equivalent volume. The significant delay in onset of ice nucleation achieved by dispensing propylene glycol in drops rather than in films is likely due to uniform dilution of the drops driven by thermocapillary flow. This transport mode is absent in the films, leading to faster dilution, and with that facilitated homogeneous nucleation, near the liquid-air interface.

  16. Antifreeze Peptides and Glycopeptides, and Their Derivatives: Potential Uses in Biotechnology

    PubMed Central

    Bang, Jeong Kyu; Lee, Jun Hyuck; Murugan, Ravichandran N.; Lee, Sung Gu; Do, Hackwon; Koh, Hye Yeon; Shim, Hye-Eun; Kim, Hyun-Cheol; Kim, Hak Jun

    2013-01-01

    Antifreeze proteins (AFPs) and glycoproteins (AFGPs), collectively called AF(G)Ps, constitute a diverse class of proteins found in various Arctic and Antarctic fish, as well as in amphibians, plants, and insects. These compounds possess the ability to inhibit the formation of ice and are therefore essential to the survival of many marine teleost fishes that routinely encounter sub-zero temperatures. Owing to this property, AF(G)Ps have potential applications in many areas such as storage of cells or tissues at low temperature, ice slurries for refrigeration systems, and food storage. In contrast to AFGPs, which are composed of repeated tripeptide units (Ala-Ala-Thr)n with minor sequence variations, AFPs possess very different primary, secondary, and tertiary structures. The isolation and purification of AFGPs is laborious, costly, and often results in mixtures, making characterization difficult. Recent structural investigations into the mechanism by which linear and cyclic AFGPs inhibit ice crystallization have led to significant progress toward the synthesis and assessment of several synthetic mimics of AFGPs. This review article will summarize synthetic AFGP mimics as well as current challenges in designing compounds capable of mimicking AFGPs. It will also cover our recent efforts in exploring whether peptoid mimics can serve as structural and functional mimics of native AFGPs. PMID:23752356

  17. H2 inhibits TNF-α-induced lectin-like oxidized LDL receptor-1 expression by inhibiting nuclear factor κB activation in endothelial cells.

    PubMed

    Song, Guohua; Tian, Hua; Liu, Jia; Zhang, Hongle; Sun, Xuejun; Qin, Shucun

    2011-09-01

    H(2) is a therapeutic antioxidant that can reduce oxidative stress. Oxidized low-density lipoprotein, which plays roles in atherosclerosis, may promote endothelial dysfunction by binding the cell-surface receptor LOX-1. LOX-1 expression can be upregulated by various stimuli, including TNF-α. Thus, we aimed to examine whether the upregulation of LOX-1 by different stimuli could be blocked by H(2) in endothelial cells. H(2) significantly abolished the upregulation of LOX-1 by different stimuli, including TNF-α, at the protein and mRNA levels. The TNF-α-induced upregulation of LOX-1 was also attenuated by the NF-κB inhibitor N-acetyl-L-cysteine. H(2) inhibited the TNF-α-induced activation of NF-κB and the phosphorylation of IκB-α. Furthermore, H(2) inhibited the expression of LOX-1 and the activation of NF-κB in apolipoprotein E knockout mice, an animal model of atherosclerosis. Thus, H(2) probably inhibits cytokine-induced LOX-1 gene expression by suppressing NF-κB activation.

  18. C-type lectin-like carbohydrate recognition of the hemolytic lectin CEL-III containing ricin-type -trefoil folds.

    PubMed

    Hatakeyama, Tomomitsu; Unno, Hideaki; Kouzuma, Yoshiaki; Uchida, Tatsuya; Eto, Seiichiro; Hidemura, Haruki; Kato, Norihisa; Yonekura, Masami; Kusunoki, Masami

    2007-12-28

    CEL-III is a Ca(2+)-dependent hemolytic lectin, isolated from the marine invertebrate Cucumaria echinata. The three-dimensional structure of CEL-III/GalNAc and CEL-III/methyl alpha-galactoside complexes was solved by x-ray crystallographic analysis. In these complexes, five carbohydrate molecules were found to be bound to two carbohydrate-binding domains (domains 1 and 2) located in the N-terminal 2/3 portion of the polypeptide and that contained beta-trefoil folds similar to ricin B-chain. The 3-OH and 4-OH of bound carbohydrate molecules were coordinated with Ca(2+) located at the subdomains 1alpha, 1gamma, 2alpha, 2beta, and 2gamma, simultaneously forming hydrogen bond networks with nearby amino acid side chains, which is similar to carbohydrate binding in C-type lectins. The binding of carbohydrates was further stabilized by aromatic amino acid residues, such as tyrosine and tryptophan, through a stacking interaction with the hydrophobic face of carbohydrates. The importance of amino acid residues in the carbohydrate-binding sites was confirmed by the mutational analyses. The orientation of bound GalNAc and methyl alpha-galactoside was similar to the galactose moiety of lactose bound to the carbohydrate-binding site of the ricin B-chain, although the ricin B-chain does not require Ca(2+) ions for carbohydrate binding. The binding of the carbohydrates induced local structural changes in carbohydrate-binding sites in subdomains 2alpha and 2beta. Binding of GalNAc also induced a slight change in the main chain structure of domain 3, which could be related to the conformational change upon binding of specific carbohydrates to induce oligomerization of the protein.

  19. The dynamics, structure, and conformational free energy of proline-containing antifreeze glycoprotein.

    PubMed Central

    Nguyen, Dat H; Colvin, Michael E; Yeh, Yin; Feeney, Robert E; Fink, William H

    2002-01-01

    Recent NMR studies of the solution structure of the 14-amino acid antifreeze glycoprotein AFGP-8 have concluded that the molecule lacks long-range order. The implication that an apparently unstructured molecule can still have a very precise function as a freezing inhibitor seems startling at first consideration. To gain insight into the nature of conformations and motions in AFGP-8, we have undertaken molecular dynamics simulations augmented with free energy calculations using a continuum solvation model. Starting from 10 different NMR structures, 20 ns of dynamics of AFGP were explored. The dynamics show that AFGP structure is composed of four segments, joined by very flexible pivots positioned at alanine 5, 8, and 11. The dynamics also show that the presence of prolines in this small AFGP structure facilitates the adoption of the poly-proline II structure as its overall conformation, although AFGP does adopt other conformations during the course of dynamics as well. The free energies calculated using a continuum solvation model show that the lowest free energy conformations, while being energetically equal, are drastically different in conformations. In other words, this AFGP molecule has many structurally distinct and energetically equal minima in its energy landscape. In addition, conformational, energetic, and hydrogen bond analyses suggest that the intramolecular hydrogen bonds between the N-acetyl group and the protein backbone are an important integral part of the overall stability of the AFGP molecule. The relevance of these findings to the mechanism of freezing inhibition is discussed. PMID:12023212

  20. FT-IR Spectra of Antifreeze Glycoproteins in Heavy Water and D2O Ice.

    NASA Astrophysics Data System (ADS)

    Tsvetkova, N. M.; Crowe, J. H.; Feeney, R. H.; Fink, W. H.; Yeh, Yin

    2000-03-01

    This work presents FT-IR studies on the antifreeze glycoprotein (AFGP)/heavy water (D2O) mixtures during freezing and melting. AFGP in the blood serum of polar fish are known to prevent ice crystal growth by a non-colligative mechanism. There are 8 known fractions of AFGP (1 8) that range in molecular mass from 33.7 to 2.6 kD respectively, each composed of alanine-alanine-threonine repeats, with a disaccharide attached to the threonine residue. The smallest peptide (AFGP-8) is structurally different from fractions 1-5 in that it contains proline substituting for alanine in certain positions. Substantial linewidth change of the D20 bending mode (ca. 1210 cm-1) was measured with solutions containing fractions 2-5 during both freezing and thawing cycles, suggesting significant coupling between protein and water molecules. At the same time, the Amide I band between 1620 and 1675 cm-1 shows that 310 helix and random coils are the main conformations of fractions 2-5 and fraction 8 in the presence of ice. In liquid state, b-sheet dominates the secondary structure of AFGP 8, whereas b-sheet and random coil are the main conformations of AFGP 2-5. These results are discussed in terms of the ability of AFGP 2-5 to affect the surface states of ice.

  1. Concentration-dependent oligomerization of an alpha-helical antifreeze polypeptide makes it hyperactive

    PubMed Central

    Mahatabuddin, Sheikh; Hanada, Yuichi; Nishimiya, Yoshiyuki; Miura, Ai; Kondo, Hidemasa; Davies, Peter L.; Tsuda, Sakae

    2017-01-01

    A supersoluble 40-residue type I antifreeze protein (AFP) was discovered in a righteye flounder, the barfin plaice (bp). Unlike all other AFPs characterized to date, bpAFP transitions from moderately-active to hyperactive with increasing concentration. At sub-mM concentrations, bpAFP bound to pyramidal planes of ice to shape it into a bi-pyramidal hexagonal trapezohedron, similarly to the other moderately-active AFPs. At mM concentrations, bpAFP uniquely underwent further binding to the whole ice crystal surface including the basal planes. The latter caused a bursting ice crystal growth normal to c-axis, 3 °C of high thermal hysteresis, and alteration of an ice crystal into a smaller lemon-shaped morphology, all of which are well-known properties of hyperactive AFPs. Analytical ultracentrifugation showed this activity transition is associated with oligomerization to form tetramer, which might be the forerunner of a naturally occurring four-helix-bundle AFP in other flounders. PMID:28211917

  2. Cyclic tensile stretch load and oxidized low density lipoprotein synergistically induce lectin-like oxidized ldl receptor-1 in cultured bovine chondrocytes, resulting in decreased cell viability and proteoglycan synthesis.

    PubMed

    Akagi, Masao; Nishimura, Shunji; Yoshida, Kohji; Kakinuma, Takumi; Sawamura, Tatsuya; Munakata, Hiroshi; Hamanishi, Chiaki

    2006-08-01

    Mechanical stimulation is known to be an essential factor in the regulation of cartilage metabolism. We tested the hypothesis that expression of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) can be modulated by cyclic tensile stretch load in chondrocytes. Cyclic loading of repeated stretch stress at 10 cycles per minute with 10 kPa of stress for 6 h induced expression of LOX-1 to 2.6 times control in cultured bovine articular chondrocytes, equivalent to the addition of 10 microg/mL oxidized low density lipoprotein (ox-LDL) (2.4 times control). Application of the cyclic load to the chondrocytes along with 10 microg/mL ox-LDL resulted in synergistically increased LOX-1 expression to 6.3 times control. Individual application of cyclic loading and 10 microg/mL ox-LDL significantly suppressed chondrocytes viability (84.6% +/- 3.4% and 80.9% +/- 3.2% of control at 24 h, respectively; n = 3; p < 0.05) and proteoglycan synthesis [81.0% +/- 7.1% and 85.7% +/- 5.2% of control at 24 h, respectively; p < 0.05 when compared with 94.6% +/- 4.6% for native-LDL (n = 3)]. Cyclic loading and 10 microg/mL ox-LDL synergistically affected cell viability and proteoglycan synthesis, which were significantly suppressed to 45.6% +/- 4.9% and 48.7% +/- 6.7% of control at 24 h, respectively (n = 3; p < 0.01 when compared with individual application of cyclic loading or 10 microg/mL ox-LDL). In this study, we demonstrated synergistic effects of cyclic tensile stretch load and ox-LDL on cell viability and proteoglycan synthesis in chondrocytes, which may be mediated through enhanced expression of LOX-1 and which has important implications in the progression of cartilage degeneration in osteoarthritis.

  3. The Snake Venom Rhodocytin from Calloselasma rhodostoma—A Clinically Important Toxin and a Useful Experimental Tool for Studies of C-Type Lectin-Like Receptor 2 (CLEC-2)

    PubMed Central

    Bruserud, Øyvind

    2013-01-01

    The snake venom, rhodocytin, from the Malayan viper, Calloselasma rhodostoma, and the endogenous podoplanin are identified as ligands for the C-type lectin-like receptor 2 (CLEC-2). The snakebites caused by Calloselasma rhodostoma cause a local reaction with swelling, bleeding and eventually necrosis, together with a systemic effect on blood coagulation with distant bleedings that can occur in many different organs. This clinical picture suggests that toxins in the venom have effects on endothelial cells and vessel permeability, extravasation and, possibly, activation of immunocompetent cells, as well as effects on platelets and the coagulation cascade. Based on the available biological studies, it seems likely that ligation of CLEC-2 contributes to local extravasation, inflammation and, possibly, local necrosis, due to microthrombi and ischemia, whereas other toxins may be more important for the distant hemorrhagic complications. However, the venom contains several toxins and both local, as well as distant, symptoms are probably complex reactions that cannot be explained by the effects of rhodocytin and CLEC-2 alone. The in vivo reactions to rhodocytin are thus examples of toxin-induced crosstalk between coagulation (platelets), endothelium and inflammation (immunocompetent cells). Very few studies have addressed this crosstalk as a part of the pathogenesis behind local and systemic reactions to Calloselasma rhodostoma bites. The author suggests that detailed biological studies based on an up-to-date methodology of local and systemic reactions to Calloselasma rhodostoma bites should be used as a hypothesis-generating basis for future functional studies of the CLEC-2 receptor. It will not be possible to study the effects of purified toxins in humans, but the development of animal models (e.g., cutaneous injections of rhodocytin to mimic snakebites) would supplement studies in humans. PMID:23594438

  4. The Activating C-type Lectin-like Receptor NKp65 Signals through a Hemi-immunoreceptor Tyrosine-based Activation Motif (hemITAM) and Spleen Tyrosine Kinase (Syk).

    PubMed

    Bauer, Björn; Wotapek, Tanja; Zöller, Tobias; Rutkowski, Emilia; Steinle, Alexander

    2017-02-24

    NKp65 is an activating human C-type lectin-like receptor (CTLR) triggering cellular cytotoxicity and cytokine secretion upon high-affinity interaction with the cognate CTLR keratinocyte-associated C-type lectin (KACL) selectively expressed by human keratinocytes. Previously, we demonstrated that NKp65-mediated cellular cytotoxicity depends on tyrosine 7, located in a cytoplasmic sequence motif of NKp65 resembling a hemi-immunoreceptor tyrosine-based activation motif (hemITAM). HemITAMs have been reported for a few activating myeloid-specific CTLRs, including Dectin-1 and CLEC-2, and consist of a single tyrosine signaling unit preceded by a triacidic motif. Upon receptor engagement, the hemITAM undergoes phosphotyrosinylation and specifically recruits spleen tyrosine kinase (Syk), initiating cellular activation. In this study, we addressed the functionality of the putative hemITAM of NKp65. We show that NKp65 forms homodimers and is phosphorylated at the hemITAM-embedded tyrosine 7 upon engagement by antibodies or KACL homodimers. HemITAM phosphotyrosinylation initiates a signaling pathway involving and depending on Syk, leading to cellular activation and natural killer (NK) cell degranulation. However, although NKp65 utilizes Syk for NK cell activation, a physical association of Syk with the NKp65 hemITAM could not be detected, unlike shown previously for the hemITAM of myeloid CTLR. Failure of NKp65 to recruit Syk is not due to an alteration of the triacidic motif, which rather affects the efficiency of hemITAM phosphotyrosinylation. In summary, NKp65 utilizes a hemITAM-like motif for cellular activation that requires Syk, although Syk appears not to be recruited to NKp65.

  5. Comparison of the solution conformation and dynamics of antifreeze glycoproteins from Antarctic fish.

    PubMed Central

    Lane, A N; Hays, L M; Tsvetkova, N; Feeney, R E; Crowe, L M; Crowe, J H

    2000-01-01

    The (1)H- and (13)C-NMR spectra of antifreeze glycoprotein fractions 1-5 from Antarctic cod have been assigned, and the dynamics have been measured using (13)C relaxation at two temperatures. The chemical shifts and absence of non-sequential (1)H-(1)H NOEs are inconsistent with a folded, compact structure. (13)C relaxation measurements show that the protein has no significant long-range order, and that the local correlation times are adequately described by a random coil model. Hydroxyl protons of the sugar residues were observed at low temperature, and the presence of exchange-mediated ROEs to the sugar indicate extensive hydration. The conformational properties of AFGP1-5 are compared with those of the previously examined 14-mer analog AFGP8, which contains proline residues in place of some alanine residues (Lane, A. N., L. M. Hays, R. E. Feeney, L. M. Crowe, and J. H. Crowe. 1998. Protein Sci. 7:1555-1563). The infrared (IR) spectra of AFGP8 and AFGP1-5 in the amide I region are quite different. The presence of a wide distribution of backbone torsion angles in AFGP1-5 leads to a rich spectrum of frequencies in the IR spectrum, as interconversion among conformational states is slow on the IR frequency time scale. However, these transitions are fast on the NMR chemical shift time scales. The restricted motions for AFGP8 may imply a narrower distribution of possible o, psi angles, as is observed in the IR spectrum. This has significance for attempts to quantify secondary structures of proteins by IR in the presence of extensive loops. PMID:10827996

  6. Antifreeze acceptability for ground-coupled heat pump ground loops in the United States

    SciTech Connect

    Den Braven, K.R.

    1998-10-01

    When designing and installing closed-loop ground-coupled heat pumps systems, it is necessary to be aware of applicable environmental regulations. Within the United States, nearly half of the states have regulations specifying or restricting the use of particular antifreezes or other fluids within the ground loop of a ground-coupled heat pump system. A number of other states have regulations pending. While all of these regulations are based on the need to preserve groundwater and/or aquifer quality, the list of acceptable antifreezes varies among those states with specified fluids. Typical antifreezes in use include ethylene glycol, propylene glycol, brines, alcohols, and potassium acetate. Each of these has its benefits and drawbacks. The status of the regulations has been determined for all of the states. An overview of the regulations is presented in this paper, along with a summary of the states` concerns.

  7. Evidence for a gamma-turn motif in antifreeze glycopeptides.

    PubMed Central

    Drewes, J A; Rowlen, K L

    1993-01-01

    Knowledge of the secondary structure of antifreeze peptides (AFPs) and glycopeptides (AFGPs) is crucial to understanding the mechanism by which these molecules inhibit ice crystal growth. A polyproline type II helix is perhaps the most widely accepted conformation for active AFGPs; however, random coil and alpha-helix conformations have also been proposed. In this report we present vibrational spectroscopic evidence that the conformation of AFGPs in solution is not random, not alpha-helical, and not polyproline type II. Comparison of AFGP amide vibrational frequencies with those observed and calculated for beta and gamma-turns in other peptides strongly suggests that AFGPs contain substantial turn structure. Computer-generated molecular models were utilized to compare gamma-turn, beta-turn, and polyproline II structures. The gamma-turn motif is consistent with observed amide frequencies and results in a molecule with planar symmetry with respect to the disaccharides. This intriguing conformation may provide new insight into the unusual properties of AFGPs. Images FIGURE 6 PMID:8241413

  8. Safe antifreeze: The real difference between ethylene glycol and propylene glycol

    SciTech Connect

    Wray, T.K.

    1995-04-01

    Antifreeze-coolants are added to the radiators of internal combustion engines to prevent freezing during the winter and boil-over during the summer. Although ethylene glycol is the most commonly used coolant, products containing propylene glycol have been used--at least, experimentally--for years. Both substances have similar characteristics; however, some manufacturers claim that antifreeze-coolants containing propylene glycol are more environmentally friendly and safer to humans and animals than ethylene glycol products. This article examines these two substances, and addresses the similarities and differences of their physical and chemical compounds, thereby enabling users to determine whether such claims are valid or merely advertising hyperbole.

  9. Heterologous expression of type I antifreeze peptide GS-5 in baker's yeast increases freeze tolerance and provides enhanced gas production in frozen dough.

    PubMed

    Panadero, Joaquin; Randez-Gil, Francisca; Prieto, Jose Antonio

    2005-12-28

    The demand for frozen-dough products has increased notably in the baking industry. Nowadays, no appropriate industrial baker's yeast with optimal gassing capacity in frozen dough is, however, available, and it is unlikely that classical breeding programs could provide significant improvements of this trait. Antifreeze proteins, found in diverse organisms, display the ability to inhibit the growth of ice, allowing them to survive at temperatures below 0 degrees C. In this study a recombinant antifreeze peptide GS-5 was expressed from the polar fish grubby sculpin (Myoxocephalus aenaeus) in laboratory and industrial baker's yeast strains of Saccharomyces cerevisiae. Production of the recombinant protein increased freezing tolerance in both strains tested. Furthermore, expression of the GS-5 encoding gene enhanced notably the gassing rate and total gas production in frozen and frozen sweet doughs. These effects are unlikely to be due to reduced osmotic damage during freezing/thawing, because recombinant cells showed growth behavior similar to that of the parent under hypermosmotic stress conditions.

  10. Effect of Antifreeze Peptide Pretreatment on Ice Crystal Size, Drip Loss, Texture, and Volatile Compounds of Frozen Carrots.

    PubMed

    Kong, Charles H Z; Hamid, Nazimah; Liu, Tingting; Sarojini, Vijayalekshmi

    2016-06-01

    Ice crystal formation is of primary concern to the frozen food industry. In this study, the effects of antifreeze peptides (AFPs) on ice crystal formation were assessed in carrot during freezing and thawing. Three synthetic analogues based on naturally occurring antifreeze peptides were used in this study. The AFPs exhibited modification of ice crystal morphology, confirming their antifreeze activity in vitro. The ability of the synthetic AFPs to minimize drip loss and preserve color, structure, texture, and volatiles of frozen carrot was evaluated using the techniques of SEM, GC-MS, and texture analysis. The results prove the potential of these AFPs to preserve the above characteristics in frozen carrot samples.

  11. Ice Growth Inhibition in Antifreeze Polypeptide Solution by Short-Time Solution Preheating

    PubMed Central

    Nishi, Naoto; Miyamoto, Takuya; Waku, Tomonori; Tanaka, Naoki; Hagiwara, Yoshimichi

    2016-01-01

    The objective of this study is to enhance the inhibition of ice growth in the aqueous solution of a polypeptide, which is inspired by winter flounder antifreeze protein. We carried out measurements on unidirectional freezing of the polypeptide solution. The thickness of the solution was 0.02 mm, and the concentration of polypeptide was varied from 0 to 2 mg/mL. We captured successive microscopic images of ice/solution interfaces, and measured the interface velocity from the locations of tips of the pectinate interface in the images. We also simultaneously measured the temperature by using a small thermocouple. The ice/solution interface temperature was defined by the temperature at the tips. It was found that the interface temperature was decreased with an increasing concentration of polypeptide. To try varying the activity of the polypeptide, we preheated the polypeptide solution and cooled it before carrying out the measurements. Preheating for 1–5 hours was found to cause a further decrease in the interface temperature. Furthermore, wider regions of solution and ice with inclined interfaces in the pectinate interface structure were observed, compared with the case where the solution was not preheated. Thus, the ice growth inhibition was enhanced by this preheating. To investigate the reason for this enhancement, we measured the conformation and aggregates of polypeptide in the solution. We also measured the local concentration of polypeptide. It was found that the polypeptide aggregates became larger as a result of preheating, although the polypeptide conformation was unchanged. These large aggregates caused both adsorption to the interface and the wide regions of supercooled solution in the pectinate interface structure. PMID:27152720

  12. Oscillations and accelerations of ice crystal growth rates in microgravity in presence of antifreeze glycoprotein impurity in supercooled water

    NASA Astrophysics Data System (ADS)

    Furukawa, Yoshinori; Nagashima, Ken; Nakatsubo, Shun-Ichi; Yoshizaki, Izumi; Tamaru, Haruka; Shimaoka, Taro; Sone, Takehiko; Yokoyama, Etsuro; Zepeda, Salvador; Terasawa, Takanori; Asakawa, Harutoshi; Murata, Ken-Ichiro; Sazaki, Gen

    2017-03-01

    The free growth of ice crystals in supercooled bulk water containing an impurity of glycoprotein, a bio-macromolecule that functions as ‘antifreeze’ in living organisms in a subzero environment, was observed under microgravity conditions on the International Space Station. We observed the acceleration and oscillation of the normal growth rates as a result of the interfacial adsorption of these protein molecules, which is a newly discovered impurity effect for crystal growth. As the convection caused by gravity may mitigate or modify this effect, secure observations of this effect were first made possible by continuous measurements of normal growth rates under long-term microgravity condition realized only in the spacecraft. Our findings will lead to a better understanding of a novel kinetic process for growth oscillation in relation to growth promotion due to the adsorption of protein molecules and will shed light on the role that crystal growth kinetics has in the onset of the mysterious antifreeze effect in living organisms, namely, how this protein may prevent fish freezing.

  13. Oscillations and accelerations of ice crystal growth rates in microgravity in presence of antifreeze glycoprotein impurity in supercooled water

    PubMed Central

    Furukawa, Yoshinori; Nagashima, Ken; Nakatsubo, Shun-ichi; Yoshizaki, Izumi; Tamaru, Haruka; Shimaoka, Taro; Sone, Takehiko; Yokoyama, Etsuro; Zepeda, Salvador; Terasawa, Takanori; Asakawa, Harutoshi; Murata, Ken-ichiro; Sazaki, Gen

    2017-01-01

    The free growth of ice crystals in supercooled bulk water containing an impurity of glycoprotein, a bio-macromolecule that functions as ‘antifreeze’ in living organisms in a subzero environment, was observed under microgravity conditions on the International Space Station. We observed the acceleration and oscillation of the normal growth rates as a result of the interfacial adsorption of these protein molecules, which is a newly discovered impurity effect for crystal growth. As the convection caused by gravity may mitigate or modify this effect, secure observations of this effect were first made possible by continuous measurements of normal growth rates under long-term microgravity condition realized only in the spacecraft. Our findings will lead to a better understanding of a novel kinetic process for growth oscillation in relation to growth promotion due to the adsorption of protein molecules and will shed light on the role that crystal growth kinetics has in the onset of the mysterious antifreeze effect in living organisms, namely, how this protein may prevent fish freezing. PMID:28262787

  14. Insect-attracting and antimicrobial properties of antifreeze for monitoring insect pests and natural enemies in stored corn.

    PubMed

    Ni, Xinzhi; Gunawan, Gunawati; Brown, Steve L; Sumner, Paul E; Ruberson, John R; Buntin, G David; Holbrook, C Corley; Lee, R Dewey; Streett, Douglas A; Throne, James E; Campbell, James F

    2008-04-01

    Insect infestations in stored grain cause extensive damage worldwide. Storage insect pests, including the Indianmeal moth, Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae); Sitophilus spp. (Coleoptera: Curculionidae); and their natural enemies [e.g., Cephalonomia tarsalis (Ashmead) (Hymenoptera: Bethylidae), and Anisopteromalus calandrae (Howard) (Hymenoptera: Pteromalidae)] inhabit a temporary, but stable ecosystem with constant environmental conditions. The objective of the present experiment was to assess the efficacy of using ethylene glycol antifreeze in combination with nutrient solutions to monitor storage insect pest and natural enemy populations in three bins of corn, Zea mays L. The treatments were deionized water, a diluted (1:5 antifreeze:water) antifreeze solution, 10% honey, 10% honey in the diluted antifreeze solution, 10% beer in the diluted antifreeze solution, 10% sucrose in the diluted antifreeze solution, and a commercial pheromone trap suspended in a 3.8-liter container filled with 300-ml of diluted antifreeze solution. The seven treatments captured storage insect pests and their natural enemies in the bins at 33-36 degrees C and 51-55% RH. The pheromone trap in the container with the diluted antifreeze captured significantly more P. interpunctella than the other treatments, but a lower percentage (7.6%) of these captures were females compared with the rest of the treatments (> 40% females). All trapping solutions also captured Sitophilus spp. and other beetle species, but the captures of the coleopteran pests were not significantly different among the seven treatments (P > 0.05). Two parasitoid wasps also were captured in the study. The number of A. calandrae was different among the seven treatments (P < 0.05), whereas the number of C. tarsalis was not different among the treatments (P > 0.05). Most A. calandrae adults were captured by the 10% honey in the diluted antifreeze, whereas the fewest were captured in the deionized water

  15. Protein-carbohydrate interactions between Lactobacillus salivarius and pig mucins.

    PubMed

    Iñiguez-Palomares, C; Jiménez-Flores, R; Vázquez-Moreno, L; Ramos-Clamont-Montfort, G; Acedo-Félix, E

    2011-10-01

    Adherence to the gastrointestinal tract is a key element desirable for many of the proposed beneficial health effects of probiotic bacteria. The aims of this study were to determine the amounts of adhesion of 3 Lactobacillus salivarius strains (Lb6, Lb9, and Lb10) to porcine small intestinal mucins and to determine whether adhesion is a function of lectin-like activities. Dot and Western blot assays were performed to investigate bacterial adhesion. Several carbohydrates and glycoproteins were evaluated to determine whether they interfered with adhesion of the Lactobacillus strains to intestinal mucins and to determine whether they had lectin-like activities. The Lb9 and Lb10 strains had greater association with piglet mucins than did those from 22- to 24-wk-old finishing pigs (P = 0.021 and 0.037, respectively), whereas the Lb6 strain adhered to both (P = 0.138). Western blot assays showed that bacterial adhesion detected piglet mucosa from the duodenum, jejunum, and ileum. In finishing pigs, the adhesion was variable throughout the gastrointestinal tract. Galactose and mannose diminished the interaction of the Lb9 and Lb10 strains in intestinal mucosa (P = 0.028 and 0.026, respectively), whereas pig gastric mucin reduced the adhesion of the Lb6 strain (P = 0.013). Adhesion of the Lb9 and Lb10 strains to intestinal mucosa was less after protease treatment (P = 0.023 and 0.018, respectively), which indicates that proteins are needed for the Lb9 and Lb10 strains to recognize mucin. The Lb6 strain also demonstrated diminished adhesion after periodate treatment (P = 0.038). From these results, we suggest that the nature of the bacterial lectin-like substance is a surface protein that loosely binds to the bacterial cell surface. All the tested strains adhered to specific targets in the small intestinal mucosa of piglets, and the bacteria had lectin-like proteins involved in this adhesion.

  16. Antifreeze Production and Cold-Tolerance in Overwintering Purple Martin Fleas, Ceratophyllus idius Jordan and Rothschild.

    DTIC Science & Technology

    1986-08-01

    survival. Can. Entomol. 110: 1167- 1205. Duman, J.G. 1977a. The role of macromolecular antifreeze in the darkling beetle , Meracantha contracta. J. Comp...Physiol. 115: 279-286. Duman, J.G. 1977b. Variations in macromolecular anti- freeze levels in larvae of the darkling beetle , Meracantha contracta. J...1967; Zachariassen and PAsche 1976). Another process has been suggested for cold-hardened Rhagium inquistor, a freeze-sensitive beetle . This insect

  17. Evaluation of anti-freeze viscosity modifier for potential external tank applications

    NASA Technical Reports Server (NTRS)

    Lynn, R. O. L.

    1981-01-01

    Viscosity modifiers and gelling agents were evaluated in combination with ethylene glycol and dimethyl sulfoxide water eutectics. Pectin and agarose are found to gel these eutectics effectively in low concentration, but the anti-freeze protection afforded by these compositions is found to be marginal in simulations of the intended applications. Oxygen vent shutters and vertical metallic surfaces were simulated, with water supplied as a spray, dropwise, and by condensation from the air.

  18. Synthesis and characterization of natural and modified antifreeze glycopeptides: glycosylated foldamers.

    PubMed

    Nagel, Lilly; Plattner, Carolin; Budke, Carsten; Majer, Zsuzsanna; DeVries, Arthur L; Berkemeier, Thomas; Koop, Thomas; Sewald, Norbert

    2011-08-01

    In Arctic and Antarctic marine regions, where the temperature declines below the colligative freezing point of physiological fluids, efficient biological antifreeze agents are crucial for the survival of polar fish. One group of such agents is classified as antifreeze glycoproteins (AFGP) that usually consist of a varying number (n = 4-55) of [AAT]( n )-repeating units. The threonine side chain of each unit is glycosidically linked to β-D: -galactosyl-(1 → 3)-α-N-acetyl-D: -galactosamine. These biopolymers can be considered as biological antifreeze foldamers. A preparative route for stepwise synthesis of AFGP allows for efficient synthesis. The diglycosylated threonine building block was introduced into the peptide using microwave-enhanced solid phase synthesis. By this versatile solid phase approach, glycosylated peptides of varying sequences and lengths could be obtained. Conformational studies of the synthetic AFGP analogs were performed by circular dichroism experiments (CD). Furthermore, the foldamers were analysed microphysically according to their inhibiting effect on ice recrystallization and influence on the crystal habit.

  19. Solution Structures, Dynamics, and Ice Growth Inhibitory Activity of Peptide Fragments Derived from an Antarctic Yeast Protein

    PubMed Central

    Asmawi, Azren A.; Rahman, Mohd Basyaruddin A.; Murad, Abdul Munir A.; Mahadi, Nor M.; Basri, Mahiran; Rahman, Raja Noor Zaliha A.; Salleh, Abu B.; Chatterjee, Subhrangsu; Tejo, Bimo A.; Bhunia, Anirban

    2012-01-01

    Exotic functions of antifreeze proteins (AFP) and antifreeze glycopeptides (AFGP) have recently been attracted with much interest to develop them as commercial products. AFPs and AFGPs inhibit ice crystal growth by lowering the water freezing point without changing the water melting point. Our group isolated the Antarctic yeast Glaciozyma antarctica that expresses antifreeze protein to assist it in its survival mechanism at sub-zero temperatures. The protein is unique and novel, indicated by its low sequence homology compared to those of other AFPs. We explore the structure-function relationship of G. antarctica AFP using various approaches ranging from protein structure prediction, peptide design and antifreeze activity assays, nuclear magnetic resonance (NMR) studies and molecular dynamics simulation. The predicted secondary structure of G. antarctica AFP shows several α-helices, assumed to be responsible for its antifreeze activity. We designed several peptide fragments derived from the amino acid sequences of α-helical regions of the parent AFP and they also showed substantial antifreeze activities, below that of the original AFP. The relationship between peptide structure and activity was explored by NMR spectroscopy and molecular dynamics simulation. NMR results show that the antifreeze activity of the peptides correlates with their helicity and geometrical straightforwardness. Furthermore, molecular dynamics simulation also suggests that the activity of the designed peptides can be explained in terms of the structural rigidity/flexibility, i.e., the most active peptide demonstrates higher structural stability, lower flexibility than that of the other peptides with lower activities, and of lower rigidity. This report represents the first detailed report of downsizing a yeast AFP into its peptide fragments with measurable antifreeze activities. PMID:23209600

  20. STUDY ON PROPERTIES OF SKID RESISTANCE ON FREEZING PAVEMENTS AND QUANTITATIVE EVALUATION METHOD OF ANTIFREEZING EFFECTS

    NASA Astrophysics Data System (ADS)

    Tanaka, Shunsuke; Takeichi, Kiyoshi; Masuyama, Yukiei; Takahashi, Naoto

    Snow and ice control in winter roads trends to be controlled by the skid friction coefficients in North America and North European countries at present, but the measurements are not necessarily easy. We studied on a simplified measurement method based on the relationship between skid friction coefficients and the bare pavement ratio (BPR) in the laboratory tests and field tests. The factors of BPR, surface textures and antifreezing materials which affect the skid friction coefficient are reviewed by a multiple linear regression analysis and a spectrum analysis, considering different freezing surfaces. These studies indicate that conclusions induced by laboratory tests could be applied to roads in service.

  1. 47 CRYOPRESERVATION OF BOVINE GERM CELL USING ANTIFREEZE POLYAMINO-ACID (CARBOXYLATED POLY-L-LYSINE).

    PubMed

    Fujikawa, T; Kubota, C; Ando, T; Imamura, S; Tokumaru, M; Yamakuchi, H; Gen, Y; Hyon, S-H

    2016-01-01

    Carboxylated poly-l-lysine (CPLL) is an ampholytic polymer compound, and it is obtained by converting 65% amino groups to carboxyl groups after synthesising ε-poly-l-lysine aqueous solution and succinic anhydride. CPLL has cryoprotective property similar to antifreeze protein, and addition of CPLL into cryopreservation medium improves the post-thaw survival rate of cells and embryos. In this research, we examined the effectiveness of CPLL as a bovine germ cell cryoprotective material. In experiment 1 (in sperm), the conventional cryopreservation medium used for control group was consisted of 6.5% (vol/vol) glycerin, and the cryopreservation medium used for CPLL group was consisted of 3.25% (vol/vol) glycerin and 0.5% CPLL (wt/vol). The post-thaw survival and motility were assessed by using Sperm Motility Analysis System (DITECT Corp., Tokyo, Japan). There was no significant difference for post-thaw survival rate and motility (control v. CPLL; 98.8% v. 96.6% and 69.7% v. 62.2%, respectively). Artificial insemination was carried out in 65 cows (control v. CPLL; 34v. 31), and the conception rate of the CPLL group was higher than that of the control group (80.6% v. 67.6%; P=0.23). In experiment 2 (embryos), the conventional cryopreservation medium used for control group was consisted of 5% (vol/vol) ethylene glycol and 6% (vol/vol) propylene glycol in PBS. In the CPLL group, 7% (wt/vol) CPLL was added to the conventional medium. In vitro fertilization embryos were cryopreserved at Day 7 and Day 8. There was no significant difference in survival rate at 0, 24, and 48h and hatched rate until 72h after thawing (control v. CPLL: 93.6% v. 93.2%, 69.0% v. 64.7%, 56.1% v. 56.3%, 12.9% v. 10.2%, respectively). Embryos obtained by superovulation treatment and in vivo fertilization at Day 7 were cryopreserved using above 2 media, and transferred non-surgically into synchronized recipient cows (1 embryo per animal). Embryo transfer (ET) was carried out in 81 cows (control v

  2. The microbubble mediated surface probe and the ice-antifreeze glycoprotein solution system

    NASA Astrophysics Data System (ADS)

    Vesenka, J. P.; Feeney, R. E.; Yeh, Y.

    1993-05-01

    Microbubble growth and its apparent "shrinkage" during the transient approach to steady-state crystal growth have been monitored by dynamic light scattering in the region immediately ahead of ice crystals growing into aqueous solutions containing dilute concentrations of macromolecules. This interfacial bubble growth occurs in the presence of a solution of globular macromolecules, and is independent of the crystal growth direction. In contrast, bubble growth becomes crystal-facet dependent when the solution contains a biological antifreeze molecule, the antifreeze glycoprotein (AFGP-4). This solution elicited an immediate, 100 x increase in bubble size above the prismatic surface of ice, followed by a gradual decrease in the averaged bubble size concomitant with a large increase in the size polydispersity. Furthermore, when the steady-state crystal growth condition is reached (in approximately one hour), the average bubble size was still ˜ 80x the size of those found in the pure ice-water system. However, when the same solution is above the basal facet, after the steady-state growth condition is attained, the microbubble diameter is unchanged from that found in the pure ice-water system. The difference in microbubble growth in the vicinity of the dynamic ice-solution interface between solutions of AFGP-4 samples and that of other molecules suggests facet-specific affinity of AFGP by ice, a condition necessary for facet-specific crystal growth inhibition.

  3. Assessment of antifreeze solutions for ground-source heat pump systems

    SciTech Connect

    Heinonen, E.W.; Tapscott, R.E.; Wildin, M.W.; Beall, A.N.

    1997-12-31

    This paper assesses the risks of using six different fluids (methanol, ethanol, aqueous potassium acetate, propylene glycol, aqueous calcium magnesium acetate, and aqueous urea) as antifreezes in ground-source heat pump (GSHP) systems. Areas assessed included fire hazard; corrosion and leakage; health hazard; environmental; detailed heat pump system analysis, resulting in predictions of annual energy use, life-cycle cost, and power plant emissions; and regulatory risk to future use. For each area, each antifreeze was rated as having either significant potential for problems, minor potential for problems, or little or no potential for problems. Propylene glycol had low risk in all areas, despite having higher energy use; potassium acetate, calcium magnesium acetate, and urea had low to medium risk in all areas except leakage, which was high for all three fluids, and corrosion, which was high for urea; ethanol and methanol had high fire risk in their pure forms (but low risk in diluted form) and corrosion problems with iron compounds. In addition, ethanol had high environmental and health risks.

  4. Identification of glycoprotein receptors within the human salivary proteome for the lectin-like BabA and SabA adhesins of Helicobacter pylori by fluorescence-based 2-D bacterial overlay.

    PubMed

    Walz, Anke; Odenbreit, Stefan; Stühler, Kai; Wattenberg, Andreas; Meyer, Helmut E; Mahdavi, Jafar; Borén, Thomas; Ruhl, Stefan

    2009-03-01

    Because gastric infection by Helicobacter pylori takes place via the oral route, possible interactions of this bacterium with human salivary proteins could occur. By using modified 1- and 2-D bacterial overlay, binding of H. pylori adhesins BabA and SabA to the whole range of salivary proteins was explored. Bound salivary receptor molecules were identified by MALDI-MS and by comparison to previously established proteome maps of whole and glandular salivas. By use of adhesin-deficient mutants, binding of H. pylori to MUC7 and gp-340 could be linked to the SabA and BabA adhesins, respectively, whereas binding to MUC5B was associated with both adhesins. Binding of H. pylori to the proline-rich glycoprotein was newly detected and assigned to BabA adhesin whereas the SabA adhesin was found to mediate binding to newly detected receptor molecules, including carbonic anhydrase VI, secretory component, heavy chain of secretory IgA1, parotid secretory protein and zinc-alpha(2)-glycoprotein. Some of these salivary glycoproteins are known to act as scavenger molecules or are involved in innate immunity whereas others might come to modify the pathogenetic properties of this organism. In general, this 2-D bacterial overlay technique represents a useful supplement in adhesion studies of bacteria with complex protein mixtures.

  5. Nanomaterials for efficiently lowering the freezing point of anti-freeze coolants.

    PubMed

    Hong, Haiping; Zheng, Yingsong; Roy, Walter

    2007-09-01

    In this paper, we report, for the first time, the effect of the lowered freezing point in a 50% water/50% anti-freeze coolant (PAC) or 50% water/50% ethylene glycol (EG) solution by the addition of carbon nanotubes and other particles. The experimental results indicated that the nano materials are much more efficient (hundreds fold) in lowering the freezing point than the regular ionic materials (e.g., NaCl). The possible explanation for this interesting phenomenon is the colligative property of fluid and relative small size of nano material. It is quite certain that the carbon nanotubes and metal oxide nano particles could be a wonderful candidate for the nano coolant application because they could not only increase the thermal conductivity, but also efficiently lower the freezing point of traditional coolants.

  6. Assessing the ability of a short fluorinated antifreeze glycopeptide and a fluorinated carbohydrate derivative to inhibit ice recrystallization.

    PubMed

    Chaytor, Jennifer L; Ben, Robert N

    2010-09-01

    A short fluorinated antifreeze glycopeptide (2) was synthesized and evaluated for ice recrystallization inhibition (IRI) activity. The activity of 2 was compared to native biological antifreeze AFGP 8 and a rationally designed C-linked AFGP analogue (OGG-Gal, 1). In addition, a simple fluorinated galactose derivative was prepared and its IRI activity was compared to non-fluorinated compounds. The results from this study suggest that the stereochemistry at the anomeric position in the carbohydrate plays a role in imparting ice recrystallization inhibition activity and that incorporation of hydrophobic groups such as fluorine atoms cause a decrease in IRI activity. These observations are consistent with the theory that fluorine atoms increase ordering of bulk water resulting in a decrease of IRI activity, supporting our previously proposed mechanism of ice recrystallization inhibition.

  7. Characterization and cloning of a Tenebrio molitor hemolymph protein with sequence similarity to insect odorant-binding proteins.

    PubMed

    Graham, L A; Tang, W; Baust, J G; Liou, Y C; Reid, T S; Davies, P L

    2001-04-27

    The yellow mealworm beetle, Tenebrio molitor, produces a number of moderately abundant low molecular weight hemolymph proteins ( approximately 12 kDa) which behave in a similar manner during purification and share antigenic epitopes. The cDNA sequence of the major component (THP12) was determined and the deduced protein sequence was found to be similar to those of insect odorant-binding proteins. Southern blot analysis suggests that at least some of the diversity in this family of proteins is encoded at the gene level. Both northern and western blot analysis indicate that THP12 is present in a variety of developmental stages and both sexes. THP12 was originally classified as an antifreeze protein, but the lack of antifreeze activity in the recombinant protein, as well as the clear separation of the antifreeze activity from THP12 following HPLC purification, has ruled out this function. The abundance of THP12, the similarity of THP12 to insect odorant-binding proteins, and the presence of hydrophobic cavities inside the protein (Rothemund et al., A new class of hexahelical insect proteins revealed as putative carriers of small hydrophobic ligands. Structure, 7 (1999) 1325-1332.) suggest that THP12 may function to carry non-water soluble compounds in the hemolymph. THP12 is also similar, particularly in structurally important regions, to other insect proteins from non-sensory tissues, suggesting the existence of a large family of carrier proteins which may perform diverse functions throughout the insect.

  8. Gliadins bind to reticulin in a lectin-like manner.

    PubMed

    Unsworth, D J; Leonard, J N; Hobday, C M; Griffiths, C E; Powles, A V; Haffenden, G P; Fry, L

    1987-01-01

    It has previously been reported that gliadins bind to reticulin in tissue sections. Three lines of evidence are reported in this study which indicate that the gliadins bind to reticulins because they are lectins which bind to sugars expressed on glycoproteins in reticulin and other sites. First, immunofluorescence studies on tissue sections showed that although gliadin binding is largely confined to areas rich in reticulin, it is, nonetheless, also seen in one or two other sites devoid of reticulin. Second, by using fluorescein-labelled lectins of known specificity, it has been shown that the areas to which gliadins bind in tissue sections (including those sites devoid of reticulin) are rich in particular sugars. Third, it has been shown that one of these sugars, alpha-D-mannose, partially inhibited gliadin binding to tissue sections.

  9. Calreticulin: one protein, one gene, many functions.

    PubMed Central

    Michalak, M; Corbett, E F; Mesaeli, N; Nakamura, K; Opas, M

    1999-01-01

    The endoplasmic reticulum (ER) plays a critical role in the synthesis and chaperoning of membrane-associated and secreted proteins. The membrane is also an important site of Ca(2+) storage and release. Calreticulin is a unique ER luminal resident protein. The protein affects many cellular functions, both in the ER lumen and outside of the ER environment. In the ER lumen, calreticulin performs two major functions: chaperoning and regulation of Ca(2+) homoeostasis. Calreticulin is a highly versatile lectin-like chaperone, and it participates during the synthesis of a variety of molecules, including ion channels, surface receptors, integrins and transporters. The protein also affects intracellular Ca(2+) homoeostasis by modulation of ER Ca(2+) storage and transport. Studies on the cell biology of calreticulin revealed that the ER membrane is a very dynamic intracellular compartment affecting many aspects of cell physiology. PMID:10567207

  10. Ancient climate change, antifreeze, and the evolutionary diversification of Antarctic fishes.

    PubMed

    Near, Thomas J; Dornburg, Alex; Kuhn, Kristen L; Eastman, Joseph T; Pennington, Jillian N; Patarnello, Tomaso; Zane, Lorenzo; Fernández, Daniel A; Jones, Christopher D

    2012-02-28

    The Southern Ocean around Antarctica is among the most rapidly warming regions on Earth, but has experienced episodic climate change during the past 40 million years. It remains unclear how ancient periods of climate change have shaped Antarctic biodiversity. The origin of antifreeze glycoproteins (AFGPs) in Antarctic notothenioid fishes has become a classic example of how the evolution of a key innovation in response to climate change can drive adaptive radiation. By using a time-calibrated molecular phylogeny of notothenioids and reconstructed paleoclimate, we demonstrate that the origin of AFGP occurred between 42 and 22 Ma, which includes a period of global cooling approximately 35 Ma. However, the most species-rich lineages diversified and evolved significant ecological differences at least 10 million years after the origin of AFGPs, during a second cooling event in the Late Miocene (11.6-5.3 Ma). This pattern indicates that AFGP was not the sole trigger of the notothenioid adaptive radiation. Instead, the bulk of the species richness and ecological diversity originated during the Late Miocene and into the Early Pliocene, a time coincident with the origin of polar conditions and increased ice activity in the Southern Ocean. Our results challenge the current understanding of the evolution of Antarctic notothenioids suggesting that the ecological opportunity that underlies this adaptive radiation is not linked to a single trait, but rather to a combination of freeze avoidance offered by AFGPs and subsequent exploitation of new habitats and open niches created by increased glacial and ice sheet activity.

  11. Computational study on the antifreeze glycoproteins as inhibitors of clathrate-hydrate formation.

    PubMed

    Cruz-Torres, Armando; Romero-Martínez, Ascención; Galano, Annia

    2008-08-04

    The ability of antifreeze glycoproteins to inhibit clathrate-hydrate formation is studied using DFT. A 5(12) cavity, dodecahedral (H(2)O)(20), and the AATA peptide are used to model the inhibitor-clathrate interaction. The presence of AATA in the vicinity of the water cavities not only leads to the formation of complexes, with different peptide/cavity ratios, but also to the deformation of the cavity and to the elongation of several of the hydrogen bonds responsible for keeping the dodecahedral (H(2)O)(20) together. The complexes are formed through hydrogen bonding between the peptides and the water cavities. The glycoproteins are expected to anchor onto the clathrate surface, blocking the access of new water molecules and preventing the incipient crystals from growing. They are also expected to weaken the clathrate structure. Amide IR bands are associated with the complexes' formation. They are significantly red-shifted in the hydrogen-bonded systems compared to isolated AATA. The amide A band is the most sensitive to hydrogen bonding. In addition a distinctive band around 3100 cm(-1) is proposed for the identification of clathrate-peptide hydrogen-bonded complexes.

  12. Inhibition of ice recrystallization and cryoprotective activity of wheat proteins in liver and pancreatic cells.

    PubMed

    Chow-Shi-Yée, Mélanie; Briard, Jennie G; Grondin, Mélanie; Averill-Bates, Diana A; Ben, Robert N; Ouellet, François

    2016-05-01

    Efficient cryopreservation of cells at ultralow temperatures requires the use of substances that help maintain viability and metabolic functions post-thaw. We are developing new technology where plant proteins are used to substitute the commonly-used, but relatively toxic chemical dimethyl sulfoxide. Recombinant forms of four structurally diverse wheat proteins, TaIRI-2 (ice recrystallization inhibition), TaBAS1 (2-Cys peroxiredoxin), WCS120 (dehydrin), and TaENO (enolase) can efficiently cryopreserve hepatocytes and insulin-secreting INS832/13 cells. This study shows that TaIRI-2 and TaENO are internalized during the freeze-thaw process, while TaBAS1 and WCS120 remain at the extracellular level. Possible antifreeze activity of the four proteins was assessed. The "splat cooling" method for quantifying ice recrystallization inhibition activity (a property that characterizes antifreeze proteins) revealed that TaIRI-2 and TaENO are more potent than TaBAS1 and WCS120. Because of their ability to inhibit ice recrystallization, the wheat recombinant proteins TaIRI-2 and TaENO are promising candidates and could prove useful to improve cryopreservation protocols for hepatocytes and insulin-secreting cells, and possibly other cell types. TaENO does not have typical ice-binding domains, and the TargetFreeze tool did not predict an antifreeze capacity, suggesting the existence of nontypical antifreeze domains. The fact that TaBAS1 is an efficient cryoprotectant but does not show antifreeze activity indicates a different mechanism of action. The cryoprotective properties conferred by WCS120 depend on biochemical properties that remain to be determined. Overall, our results show that the proteins' efficiencies vary between cell types, and confirm that a combination of different protection mechanisms is needed to successfully cryopreserve mammalian cells.

  13. Stabilization of supercooled fluids by thermal hysteresis proteins.

    PubMed Central

    Wilson, P W; Leader, J P

    1995-01-01

    It has been reported that thermal hysteresis proteins found in many cold-hardy, freeze-avoiding arthropods stabilize their supercooled body fluids. We give evidence that fish antifreeze proteins, which also produce thermal hysteresis, bind to and reduce the efficiency of heterogenous nucleation sites, rather than binding to embryonic ice nuclei. We discuss both possible mechanisms for stabilization of supercooled body fluids and also describe a new method for measuring and defining the supercooling point of small volumes of liquid. PMID:7612853

  14. Ancient climate change, antifreeze, and the evolutionary diversification of Antarctic fishes

    PubMed Central

    Near, Thomas J.; Dornburg, Alex; Kuhn, Kristen L.; Eastman, Joseph T.; Pennington, Jillian N.; Patarnello, Tomaso; Zane, Lorenzo; Fernández, Daniel A.; Jones, Christopher D.

    2012-01-01

    The Southern Ocean around Antarctica is among the most rapidly warming regions on Earth, but has experienced episodic climate change during the past 40 million years. It remains unclear how ancient periods of climate change have shaped Antarctic biodiversity. The origin of antifreeze glycoproteins (AFGPs) in Antarctic notothenioid fishes has become a classic example of how the evolution of a key innovation in response to climate change can drive adaptive radiation. By using a time-calibrated molecular phylogeny of notothenioids and reconstructed paleoclimate, we demonstrate that the origin of AFGP occurred between 42 and 22 Ma, which includes a period of global cooling approximately 35 Ma. However, the most species-rich lineages diversified and evolved significant ecological differences at least 10 million years after the origin of AFGPs, during a second cooling event in the Late Miocene (11.6–5.3 Ma). This pattern indicates that AFGP was not the sole trigger of the notothenioid adaptive radiation. Instead, the bulk of the species richness and ecological diversity originated during the Late Miocene and into the Early Pliocene, a time coincident with the origin of polar conditions and increased ice activity in the Southern Ocean. Our results challenge the current understanding of the evolution of Antarctic notothenioids suggesting that the ecological opportunity that underlies this adaptive radiation is not linked to a single trait, but rather to a combination of freeze avoidance offered by AFGPs and subsequent exploitation of new habitats and open niches created by increased glacial and ice sheet activity. PMID:22331888

  15. Taste responses of dogs to ethylene glycol, propylene glycol, and ethylene glycol-based antifreeze.

    PubMed

    Marshall, D A; Doty, R L

    1990-12-15

    Although it is widely believed that ethylene glycol-based antifreeze (AF) is an attractive tastant to dogs and other animals, empirical data on this point are not available. In experiment 1, we examined the propensity of 178 adult mixed-breed dogs to approach, sniff, and lick a concentration of AF commonly used in automotive cooling systems (50%). Despite the fact that most of the dogs approached and sniffed the AF in these 5-minute tests, only 9% initiated lick responses and most of these were brief and not followed by additional licking. In experiment 2, the lick responses of five gastric-cannulated dogs to aqueous solutions of 20% sucrose, 50% ethylene glycol, 50% propylene glycol, water, and 50% AF were examined in 14-minute tests before and after periods of food and water deprivation. Under the latter conditions, 2 of the 5 dogs drank amounts of ethylene glycol that would have been lethal to uncannulated dogs. None of the five dogs drank potentially lethal amounts of AF. The preference order for these tastants was sucrose greater than water greater than ethylene glycol greater than AF = propylene glycol. Although these findings question the general belief that AF is highly palatable to most dogs, they do imply that large individual differences in responsiveness exist and that AF ingestion is likely influenced by motivational state. Furthermore, they suggest the possibility that unpleasant-tasting additives could be successfully developed to eliminate the ingestion of AF, because the initial attractiveness of AF is relatively low. Such additives would have to be stable in vehicular cooling systems and not adversely affect the functional aspects of AF performance.

  16. Ice growth in supercooled solutions of a biological "antifreeze", AFGP 1-5: an explanation in terms of adsorption rate for the concentration dependence of the freezing point.

    PubMed

    Knight, C A; DeVries, A L

    2009-07-21

    It is widely accepted, and we agree, that the lowering of the temperature at which ice can grow in a water solution of one of the biological antifreezes is a result of adsorption of the antifreeze molecules at the ice surface. However, how this can produce a well-defined "freezing point" that varies with the solution concentration has remained problematical. The results of a series of measurements of ice growing in supercooled solutions of an effective antifreeze are reported and interpreted in terms of this fundamental problem. It seemed that the solution of the problem would have to rely upon adsorption rate, because that appeared to be the only way for the concentration in solution to be so important. The crystal growth results are most unusual, and appear to confirm this. The growth rates over a wide range of antifreeze concentration in solution (about 0.05 to 9 mg ml(-1)) are zero from the thermodynamic freezing point down to the "non-equilibrium" freezing point, where there is a very sudden increase to a plateau value that then remains about constant as the supercooling is increased by about 2 degrees C. The plateau values of growth rate are faster than those from pure water at the lower-supercooling ends of the plateaus, but slower at higher supercooling, until the growth rate starts rising toward that from pure water. These plateau values of growth rate increase markedly with increasing concentration of the antifreeze in solution. Along with these changes there are complex changes in the growth orientations, from c-axis spicules in the plateaus to those more characteristic of growth from pure water at greater supercooling. We conclude that the non-equilibrium freezing point is determined by the adsorption rate. It is the warmest temperature at which the ice growth rate on the basal plane (where the antifreeze does not adsorb) is fast enough to prevent the area of basal face on a growing ice crystal from becoming too small to grow, which is determined in

  17. Characterization of the restricted rotation of the dimethyl groups in chemically N-terminal 13C-labeled antifreeze glycoproteins: A temperature-dependent study in water to ice through the supercooled state

    NASA Astrophysics Data System (ADS)

    Krishnan, V. V.; Lau, Edmond Y.; Tsvetkova, Nelly M.; Feeney, Robert E.; Fink, William H.; Yeh, Yin

    2005-07-01

    Site-specific chemical modification, especially with isotopically enriched groups, allows one to study the structure and dynamics of proteins for which uniform enrichment is difficult. When the N-terminal alanine in antifreeze glycoprotein (AFGP) is replaced with an N,N-dimethyl alanine the methyl groups show signatures of slow rotation about the C-N bond. In order to separate the local dynamics of the N-terminus from the overall protein dynamics, we present a complete characterization of this dynamics. Temperature-dependent nuclear magnetic-resonance experiments from room temperature to subzero temperatures, including the supercooled state and in the presence of ice, are presented. Quantum chemical calculations are also performed on a localized N-terminus of the AFGP. Our results show that in the solution state at room temperature and in the super cooled regime, the dimethyl groups undergo a slow, restricted rotation with an unequal distribution of population between two major conformations. At lower temperatures in the presence of ice, the dynamics become much more complex due to freezing out of several conformational states. Based on these results, we conclude that the segmental dynamics of the N-terminus are local to the first residue and do not affect the overall dynamics of the protein.

  18. Anaplasma phagocytophilum induces Ixodes scapularis ticks to express an antifreeze glycoprotein gene that enhances their survival in the cold

    PubMed Central

    Neelakanta, Girish; Sultana, Hameeda; Fish, Durland; Anderson, John F.; Fikrig, Erol

    2010-01-01

    In the United States, Ixodes scapularis ticks overwinter in the Northeast and Upper Midwest and transmit the agent of human granulocytic anaplasmosis, Anaplasma phagocytophilum, among other pathogens. We now show that the presence of A. phagocytophilum in I. scapularis ticks increases their ability to survive in the cold. We identified an I. scapularis antifreeze glycoprotein, designated IAFGP, and demonstrated via RNAi knockdown studies the importance of IAFGP for the survival of I. scapularis ticks in a cold environment. Transfection studies also show that IAFGP increased the viability of yeast cells subjected to cold temperature. Remarkably, A. phagocytophilum induced the expression of iafgp, thereby increasing the cold tolerance and survival of I. scapularis. These data define a molecular basis for symbiosis between a human pathogenic bacterium and its arthropod vector and delineate what we believe to be a new pathway that may be targeted to alter the life cycle of this microbe and its invertebrate host. PMID:20739755

  19. Thermal hysteresis proteins.

    PubMed

    Barrett, J

    2001-02-01

    Extreme environments present a wealth of biochemical adaptations. Thermal hysteresis proteins (THPs) have been found in vertebrates, invertebrates, plants, bacteria and fungi and are able to depress the freezing point of water (in the presence of ice crystals) in a non-colligative manner by binding to the surface of nascent ice crystals. The THPs comprise a disparate group of proteins with a variety of tertiary structures and often no common sequence similarities or structural motifs. Different THPs bind to different faces of the ice crystal, and no single mechanism has been proposed to account for THP ice binding affinity and specificity. Experimentally THPs have been used in the cryopreservation of tissues and cells and to induce cold tolerance in freeze susceptible organisms. THPs represent a remarkable example of parallel and convergent evolution with different proteins being adapted for an anti-freeze role.

  20. Isolation and characterisation of sericin antifreeze peptides and molecular dynamics modelling of their ice-binding interaction.

    PubMed

    Wu, Jinhong; Rong, Yuzhi; Wang, Zhengwu; Zhou, Yanfu; Wang, Shaoyun; Zhao, Bo

    2015-05-01

    This study aimed to isolate and characterise a novel sericin antifreeze peptide and investigate its ice-binding molecular mechanism. The thermal hysteresis activity of ice-binding sericin peptides (I-SP) was measured and their activity reached as high as 0.94 °C. A P4 fraction, with high hypothermia protective activity and inhibition activity of ice recrystallisation, was obtained from I-SP, and a purified sericin peptide, named SM-AFP, with the sequence of TTSPTNVSTT and a molecular weight of 1009.50 Da was then isolated from the P4 fraction. Treatment of Lactobacillus delbrueckii Subsp. bulgaricus LB340 LYO with 100 μg/ml synthetic SM-AFP led to 1.4-fold increased survival (p < 0.05). Finally, an SM-AFP/ice binding model was constructed and results of molecular dynamics simulation suggested that the binding of SM-AFP with ice and prevention of ice crystal growth could be attributed to hydrogen bond formation, hydrophobic interaction and non-bond interactions. Sericin peptides could be developed into beneficial cryoprotectants and used in frozen food processing.

  1. Relationship of amino acid composition and molecular weight of antifreeze glycopeptides to non-colligative freezing point depression.

    PubMed

    Schrag, J D; O'Grady, S M; DeVries, A L

    1982-08-06

    Many polar fishes synthesize a group of eight glycopeptides that exhibit a non-colligative lowering of the freezing point of water. These glycopeptides range in molecular weight between 2600 and 33 700. The largest glycopeptides [1-5] lower the freezing point more than the small ones on a weight basis and contain only two amino acids, alanine and threonine, with the disaccharide galactose-N-acetyl-galactosamine attached to threonine. The small glycopeptides, 6, 7, and 8, also lower the freezing point and contain proline, which periodically substitutes for alanine. Glycopeptides with similar antifreeze properties isolated from the saffron cod and the Atlantic tomcod contain an additional amino acid, arginine, which substitutes for threonine in glycopeptide 6. In this study we address the question of whether differences in amino acid composition or molecular weight between large and small glycopeptides are responsible for the reduced freezing point depressing capability of the low molecular weight glycopeptides. The results indicate that the degree of amino acid substitutions that occur in glycopeptides 6-8 do not have a significant effect on the unusual freezing point lowering and that the observed decrease in freezing point depression with smaller glycopeptides can be accounted for on the basis of molecular weight.

  2. Influence of surface groups of proteins on water studied by freezing/thawing hysteresis and infrared spectroscopy.

    PubMed

    Zelent, Bogumil; Bryan, Michael A; Sharp, Kim A; Vanderkooi, Jane M

    2009-05-01

    The influence of proteins and solutes on hysteresis of freezing and melting of water was measured by infrared (IR) spectroscopy. Of the solutes examined, poly-L-arginine and flounder antifreeze protein produced the largest freezing point depression of water, with little effect on the melting temperature. Poly-L-lysine, poly-L-glutamate, cytochrome c and bovine serum albumin had less effect on the freezing of water. Small compounds used to mimic non-polar (trimethylamine N-oxide, methanol), positively charged (guanidinium chloride, NH(4)Cl, urea) and negatively charged (Na acetate) groups on protein surfaces were also examined. These molecules and ions depress water's freezing point and the melting profiles became broad. Since infrared absorption measures both bulk solvent and solvent bound to the solutes, this result is consistent with solutes interacting with liquid water. The amide I absorption bands of antifreeze protein and poly-L-arginine do not detectably change with the phase transition of water. An interpretation is that the antifreeze protein and poly-L-arginine order liquid water such that the water around the group is ice-like.

  3. Mimicking biopolymers on a molecular scale: nano(bio)technology based on engineered proteins.

    PubMed

    Grunwald, Ingo; Rischka, Klaus; Kast, Stefan M; Scheibel, Thomas; Bargel, Hendrik

    2009-05-13

    Proteins are ubiquitous biopolymers that adopt distinct three-dimensional structures and fulfil a multitude of elementary functions in organisms. Recent systematic studies in molecular biology and biotechnology have improved the understanding of basic functional and architectural principles of proteins, making them attractive candidates as concept generators for technological development in material science, particularly in biomedicine and nano(bio)technology. This paper highlights the potential of molecular biomimetics in mimicking high-performance proteins and provides concepts for applications in four case studies, i.e. spider silk, antifreeze proteins, blue mussel adhesive proteins and viral ion channels.

  4. The effects of antifreeze peptide III (AFP) and insulin transferrin selenium (ITS) on cryopreservation of chimpanzee (Pan troglodytes) spermatozoa.

    PubMed

    Younis, A I; Rooks, B; Khan, S; Gould, K G

    1998-01-01

    We investigated the effects of antifreeze peptides (AFP) and insulin transferrin selenium (ITS) on the motility and membrane integrity of chimpanzee (Pan troglodytes) spermatozoa after chilling (0-5 degrees C) and thawing. The effects of three thawing procedures, in the presence or absence of AFP and ITS, on sperm motility and on the status of the plasma membrane and acrosome were also examined. During chilling, AFP and ITS seem mildly cytotoxic, as the progressive motility and velocity (curvilinear and straight line) declined significantly at AFP concentrations of 1, 10, and 100 microg/ml and at ITS concentrations of 1 and 10 microg/ml. However, at a concentration of 100 microg/ml, ITS was able to protect sperm during short-term hypothermic storage. Addition of AFP or ITS at 100 microg/ml to test egg yolk-glycerol extender during freezing significantly (P < 0.05) increased postthaw motility, plasma membrane integrity, and acrosome integrity. The mean (+/-SE) motility recovery rate increased from 28.9 +/- 3.9%, for the untreated control, to 59.2 +/- 5.8% and 67.8 +/- 7.4%, for ITS and AFP, respectively. The effects of the thawing procedure were influenced by the presence of AFP during the freezing cycle. An improved motility recovery rate of 67 +/- 4.2% was obtained when chimpanzee sperm frozen in test egg yolk-glycerol extender supplemented with AFP were thawed rapidly at 37 degrees C, compared to 47 +/- 5.2% and 44 +/- 8.2% for slow (23 degrees C) and ultrarapid (75 degrees C) thawing, respectively. The motility recovery after thawing of ITS-treated semen at 23 degrees C, 37 degrees C, or 75 degrees C was not significantly different. Semen frozen without AFP or ITS and thawed at 75 degrees C was seriously (P < 0.05) damaged. This study provides evidence that AFP- or ITS-supplemented semen extender improves postthaw sperm motility in the chimpanzee.

  5. Enhanced inactivation of avian influenza virus at -20°C by disinfectants supplemented with calcium chloride or other antifreeze agents.

    PubMed

    Guan, Jiewen; Chan, Maria; Brooks, Brian W; Rohonczy, Elizabeth

    2015-10-01

    Avian influenza outbreaks have occurred during winter months, and effective disinfection of poultry premises at freezing temperatures is needed. The commercial disinfectants Virkon and Accel, supplemented with an antifreeze agent [propylene glycol (PG), methanol (MeOH), or calcium chloride (CaCl₂)], were evaluated for their effectiveness in killing avian influenza virus (AIV) at -20°C or 21°C. An AIV suspension was applied to stainless steel disks, air-dried, and covered with a disinfectant or antifreeze agent for 5 to 30 min. Virkon (2%) and Accel (6.25%) with 30% PG, 20% MeOH, or 20% CaCl₂ inactivated 6 log₁₀ AIV within 5 min at -20°C and 21°C. At these temperatures PG and MeOH alone did not kill AIV, but the 20% CaCl₂ solution alone inactivated 5 log10 AIV within 10 min. The results suggested that CaCl₂ is potentially useful to enhance the effectiveness of disinfection of poultry facilities after outbreaks of AIV infection in warm and cold seasons.

  6. Enhanced inactivation of avian influenza virus at −20°C by disinfectants supplemented with calcium chloride or other antifreeze agents

    PubMed Central

    Guan, Jiewen; Chan, Maria; Brooks, Brian W.; Rohonczy, Elizabeth

    2015-01-01

    Avian influenza outbreaks have occurred during winter months, and effective disinfection of poultry premises at freezing temperatures is needed. The commercial disinfectants Virkon and Accel, supplemented with an antifreeze agent [propylene glycol (PG), methanol (MeOH), or calcium chloride (CaCl2)], were evaluated for their effectiveness in killing avian influenza virus (AIV) at −20°C or 21°C. An AIV suspension was applied to stainless steel disks, air-dried, and covered with a disinfectant or antifreeze agent for 5 to 30 min. Virkon (2%) and Accel (6.25%) with 30% PG, 20% MeOH, or 20% CaCl2 inactivated 6 log10 AIV within 5 min at −20°C and 21°C. At these temperatures PG and MeOH alone did not kill AIV, but the 20% CaCl2 solution alone inactivated 5 log10 AIV within 10 min. The results suggested that CaCl2 is potentially useful to enhance the effectiveness of disinfection of poultry facilities after outbreaks of AIV infection in warm and cold seasons. PMID:26424918

  7. Protein Carbamylation and Cardiovascular Disease

    PubMed Central

    Verbrugge, Frederik H.; Tang, W.H. Wilson; Hazen, Stanley L.

    2015-01-01

    Carbamylation constitutes a posttranslational modification of proteins or amino acids and results from different pathways in vivo. First is the non-enzymatic reaction between isocyanic acid, a decomposition product of urea, and either the N-terminus or ε-amino group of lysine residues. Isocyanic acid levels, while low in vivo, are in equilibrium with urea, and are thus increased in chronic and end-stage renal diseases. An alternative pathway involves the leukocyte haem protein myeloperoxidase, which catalyses the oxidation of thiocyanate in the presence of hydrogen peroxide, producing isocyanate at inflammation sites. Notably, plasma thiocyanate levels are increased in smokers, and leukocyte-driven protein carbamylation occurs both within human and animal atherosclerotic plaques, as well as on plasma proteins. Protein carbamylation is considered a hallmark of molecular aging and is implicated in many pathological conditions. Recently, it has been shown that carbamylated low-density lipoprotein (LDL) induces endothelial dysfunction via lectin-like-oxidized LDL receptor-1 activation and increased reactive oxygen species production, leading to endothelial nitric oxide synthase uncoupling. Moreover, carbamylated LDL harbours atherogenic activities, including both binding to macrophage scavenger receptors inducing cholesterol accumulation and foam cell formation, as well as promoting vascular smooth muscle proliferation. In contrast, high-density lipoprotein loses its anti-apoptotic activity after carbamylation, contributing to endothelial cell death. In addition to involvement in atherogenesis, protein carbamylation levels have emerged as a particularly strong predictor of both prevalent and incident cardiovascular disease risk. Recent studies also suggest that protein carbamylation may serve as a potential therapeutic target for the prevention of atherosclerotic heart disease. PMID:26061545

  8. The N-terminal domain of thrombomodulin sequesters high-mobility group-B1 protein, a novel antiinflammatory mechanism

    PubMed Central

    Abeyama, Kazuhiro; Stern, David M.; Ito, Yuji; Kawahara, Ko-ichi; Yoshimoto, Yasushi; Tanaka, Motoyuki; Uchimura, Tomonori; Ida, Nobuo; Yamazaki, Yoshiaki; Yamada, Shingo; Yamamoto, Yasuhiko; Yamamoto, Hiroshi; Iino, Satoshi; Taniguchi, Noboru; Maruyama, Ikuro

    2005-01-01

    Thrombomodulin (TM) is an endothelial anticoagulant cofactor that promotes thrombin-mediated formation of activated protein C (APC). We have found that the N-terminal lectin-like domain (D1) of TM has unique antiinflammatory properties. TM, via D1, binds high-mobility group-B1 DNA-binding protein (HMGB1), a factor closely associated with necrotic cell damage following its release from the nucleus, thereby preventing in vitro leukocyte activation, in vivo UV irradiation–induced cutaneous inflammation, and in vivo lipopolysaccharide-induced lethality. Our data also demonstrate antiinflammatory properties of a peptide spanning D1 of TM and suggest its therapeutic potential. These findings highlight a novel mechanism, i.e., sequestration of mediators, through which an endothelial cofactor, TM, suppresses inflammation quite distinctly from its anticoagulant cofactor activity, thereby preventing the interaction of these mediators with cell surface receptors on effector cells in the vasculature. PMID:15841214

  9. Proteins.

    ERIC Educational Resources Information Center

    Doolittle, Russell F.

    1985-01-01

    Examines proteins which give rise to structure and, by virtue of selective binding to other molecules, make genes. Binding sites, amino acids, protein evolution, and molecular paleontology are discussed. Work with encoding segments of deoxyribonucleic acid (exons) and noncoding stretches (introns) provides new information for hypotheses. (DH)

  10. Interstellar Antifreeze: Ethylene Glycol

    NASA Technical Reports Server (NTRS)

    Hollis, J. M.; Lovas, F. J.; Jewell, P. R.; Coudert, L. H.

    2002-01-01

    Interstellar ethylene glycol (HOCH2CH2,OH) has been detected in emission toward the Galactic center source Sagittarius B2(N-LMH) by means of several millimeter-wave rotational torsional transitions of its lowest energy conformer. The types and kinds of molecules found to date in interstellar clouds suggest a chemistry that favors aldehydes and their corresponding reduced alcohols-e.g., formaldehyde (H2CO)/methanol (CH3OH), acetaldehyde (CH3CHO)/ethanol (CH3CH2OH). Similarly, ethylene glycol is the reduced alcohol of glycolaldehyde (CH2OHCHO), which has also been detected toward Sgr B2(N-LMH). While there is no consensus as to how any such large complex molecules are formed in the interstellar clouds, atomic hydrogen (H) and carbon monoxide (CO) could form formaldehyde on grain surfaces, but such surface chemistry beyond that point is uncertain. However, laboratory experiments have shown that the gas-phase reaction of atomic hydrogen (H) and solid-phase CO at 10-20 K can produce formaldehyde and methanol and that alcohols and other complex molecules can be synthesized from cometary ice analogs when subject to ionizing radiation at 15 K. Thus, the presence of aldehyde/ reduced alcohol pairs in interstellar clouds implies that such molecules are a product of a low-temperature chemistry on grain surfaces or in grain ice mantles. This work suggests that aldehydes and their corresponding reduced alcohols provide unique observational constraints on the formation of complex interstellar molecules.

  11. Protein

    MedlinePlus

    ... Search for: Harvard T.H. Chan School of Public Health Email People Departments Calendar Careers Give my.harvard ... Nutrition Source Harvard T.H. Chan School of Public Health > The Nutrition Source > What Should I Eat? > Protein ...

  12. Protein

    MedlinePlus

    ... Go lean with protein. • Choose lean meats and poultry. Lean beef cuts include round steaks (top loin, ... main dishes. • Use nuts to replace meat or poultry, not in addition to meat or poultry (i. ...

  13. Ice-binding proteins that accumulate on different ice crystal planes produce distinct thermal hysteresis dynamics

    PubMed Central

    Drori, Ran; Celik, Yeliz; Davies, Peter L.; Braslavsky, Ido

    2014-01-01

    Ice-binding proteins that aid the survival of freeze-avoiding, cold-adapted organisms by inhibiting the growth of endogenous ice crystals are called antifreeze proteins (AFPs). The binding of AFPs to ice causes a separation between the melting point and the freezing point of the ice crystal (thermal hysteresis, TH). TH produced by hyperactive AFPs is an order of magnitude higher than that produced by a typical fish AFP. The basis for this difference in activity remains unclear. Here, we have compared the time dependence of TH activity for both hyperactive and moderately active AFPs using a custom-made nanolitre osmometer and a novel microfluidics system. We found that the TH activities of hyperactive AFPs were time-dependent, and that the TH activity of a moderate AFP was almost insensitive to time. Fluorescence microscopy measurement revealed that despite their higher TH activity, hyperactive AFPs from two insects (moth and beetle) took far longer to accumulate on the ice surface than did a moderately active fish AFP. An ice-binding protein from a bacterium that functions as an ice adhesin rather than as an antifreeze had intermediate TH properties. Nevertheless, the accumulation of this ice adhesion protein and the two hyperactive AFPs on the basal plane of ice is distinct and extensive, but not detectable for moderately active AFPs. Basal ice plane binding is the distinguishing feature of antifreeze hyperactivity, which is not strictly needed in fish that require only approximately 1°C of TH. Here, we found a correlation between the accumulation kinetics of the hyperactive AFP at the basal plane and the time sensitivity of the measured TH. PMID:25008081

  14. Ice-binding proteins that accumulate on different ice crystal planes produce distinct thermal hysteresis dynamics.

    PubMed

    Drori, Ran; Celik, Yeliz; Davies, Peter L; Braslavsky, Ido

    2014-09-06

    Ice-binding proteins that aid the survival of freeze-avoiding, cold-adapted organisms by inhibiting the growth of endogenous ice crystals are called antifreeze proteins (AFPs). The binding of AFPs to ice causes a separation between the melting point and the freezing point of the ice crystal (thermal hysteresis, TH). TH produced by hyperactive AFPs is an order of magnitude higher than that produced by a typical fish AFP. The basis for this difference in activity remains unclear. Here, we have compared the time dependence of TH activity for both hyperactive and moderately active AFPs using a custom-made nanolitre osmometer and a novel microfluidics system. We found that the TH activities of hyperactive AFPs were time-dependent, and that the TH activity of a moderate AFP was almost insensitive to time. Fluorescence microscopy measurement revealed that despite their higher TH activity, hyperactive AFPs from two insects (moth and beetle) took far longer to accumulate on the ice surface than did a moderately active fish AFP. An ice-binding protein from a bacterium that functions as an ice adhesin rather than as an antifreeze had intermediate TH properties. Nevertheless, the accumulation of this ice adhesion protein and the two hyperactive AFPs on the basal plane of ice is distinct and extensive, but not detectable for moderately active AFPs. Basal ice plane binding is the distinguishing feature of antifreeze hyperactivity, which is not strictly needed in fish that require only approximately 1°C of TH. Here, we found a correlation between the accumulation kinetics of the hyperactive AFP at the basal plane and the time sensitivity of the measured TH.

  15. Purification and characterization of a novel immunomodulatory protein from the medicinal mushroom Trametes versicolor.

    PubMed

    Li, Feng; Wen, Huaan; Zhang, Yongjie; Aa, Min; Liu, Xingzhong

    2011-04-01

    Bioactive proteins represent an important group of functional agents in medicinal mushrooms. Trametes versicolor (L.) Lloyd is a mushroom frequently used in traditional Chinese medicine for its anti-tumor and immunomodulatory activities. A new immunomodulatory protein from T. versicolor, named TVC, was purified by ammonium sulfate precipitation, ion-exchange chromatography and gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified protein revealed a single band with a molecular weight of 15.0 kD. Native polyacrylamide gel analysis revealed a band at 30 kD, indicating that TVC exists in solution as a homodimer. Isoelectric focusing showed that TVC was an acidic protein with an isoelectric point of 4.0. TVC was found to lack carbohydrate modifications (based on periodic acid/Schiff staining) and it does not agglutinate mouse red blood cells, suggesting that TVC is not a lectin-like protein. Biological activity assays demonstrated that TVC can enhance the proliferation of splenocytes, while it has no stimulatory effects on CD4(+) and CD8(+) T cells. TVC markedly increases the proliferation of human peripheral blood lymphocytes in a dose-dependent manner and enhances the production of both nitric oxide and tumor necrosis factor-alpha by lipopolysaccharide-induced murine macrophages. The results indicate that TVC is an immunostimulant that can boost immune response. Comparison of the N-terminal amino acid residues and mass spectrometry results with the protein database revealed no homologous proteins.

  16. Ice-active proteins from New Zealand snow tussocks, Chionochloa macra AND C. rigida.

    PubMed

    Wharton, D A; Selvanesan, L; Marshall, C J

    2010-01-01

    The ice active protein profile of New Zealand snow tussocks Chionochloa macra and C. rigida consisted of ice nucleation activity but no antifreeze or recrystallization inhibition activity. The ice nucleation activity was similar in the two species, despite them being collected at different altitudes and at different times. The activity is intrinsic to the plant and is associated with the surface of the leaves. Snow tussocks collect water from fog. Nucleation sites on the surface of their leaves may aid the efficiency of this process.

  17. Cloning of fish enzymes and other fish protein genes.

    PubMed

    Macouzet, M; Simpson, B K; Lee, B H

    1999-01-01

    Fish metabolism needs special enzymes that have maximum activity at very different conditions than their mammalian counterparts. Due to the differences in activity, these enzymes, especially cold-adapted proteases, could be used advantageously for the production of some foods. In addition to the enzymes, this review describes some other unique fish polypeptides such as antifreeze proteins, fluorescent proteins, antitumor peptides, antibiotics, and hormones, that have already been cloned and used in food processing, genetic engineering, medicine, and aquaculture. Recombinant DNA technology, which allows these biological molecules to be cloned and overexpressed in microorganisms is also described, highlighting innovative applications. The expected impact of cloning fish proteins in different fields of technology is discussed.

  18. Latent Ice Recrystallization Inhibition Activity in Nonantifreeze Proteins: Ca2+-Activated Plant Lectins and Cation-Activated Antimicrobial Peptides.

    PubMed

    Mitchell, Daniel E; Gibson, Matthew I

    2015-10-12

    Organisms living in polar regions have evolved a series of antifreeze (glyco) proteins (AFGPs) to enable them to survive by modulating the structure of ice. These proteins have huge potential for use in cellular cryopreservation, ice-resistant surfaces, frozen food, and cryosurgery, but they are limited by their relatively low availability and questions regarding their mode of action. This has triggered the search for biomimetic materials capable of reproducing this function. The identification of new structures and sequences capable of inhibiting ice growth is crucial to aid our understanding of these proteins. Here, we show that plant c-type lectins, which have similar biological function to human c-type lectins (glycan recognition) but no sequence homology to AFPs, display calcium-dependent ice recrystallization inhibition (IRI) activity. This IRI activity can be switched on/off by changing the Ca2+ concentration. To show that more (nonantifreeze) proteins may exist with the potential to display IRI, a second motif was considered, amphipathicity. All known AFPs have defined hydrophobic/hydrophilic domains, rationalizing this choice. The cheap, and widely used, antimicrobial Nisin was found to have cation-dependent IRI activity, controlled by either acid or addition of histidine-binding ions such as zinc or nickel, which promote its amphipathic structure. These results demonstrate a new approach in the identification of antifreeze protein mimetic macromolecules and may help in the development of synthetic mimics of AFPs.

  19. Latent Ice Recrystallization Inhibition Activity in Nonantifreeze Proteins: Ca2+-Activated Plant Lectins and Cation-Activated Antimicrobial Peptides

    PubMed Central

    2015-01-01

    Organisms living in polar regions have evolved a series of antifreeze (glyco) proteins (AFGPs) to enable them to survive by modulating the structure of ice. These proteins have huge potential for use in cellular cryopreservation, ice-resistant surfaces, frozen food, and cryosurgery, but they are limited by their relatively low availability and questions regarding their mode of action. This has triggered the search for biomimetic materials capable of reproducing this function. The identification of new structures and sequences capable of inhibiting ice growth is crucial to aid our understanding of these proteins. Here, we show that plant c-type lectins, which have similar biological function to human c-type lectins (glycan recognition) but no sequence homology to AFPs, display calcium-dependent ice recrystallization inhibition (IRI) activity. This IRI activity can be switched on/off by changing the Ca2+ concentration. To show that more (nonantifreeze) proteins may exist with the potential to display IRI, a second motif was considered, amphipathicity. All known AFPs have defined hydrophobic/hydrophilic domains, rationalizing this choice. The cheap, and widely used, antimicrobial Nisin was found to have cation-dependent IRI activity, controlled by either acid or addition of histidine-binding ions such as zinc or nickel, which promote its amphipathic structure. These results demonstrate a new approach in the identification of antifreeze protein mimetic macromolecules and may help in the development of synthetic mimics of AFPs. PMID:26407233

  20. Crystal Structure of Ribosome-Inactivating Protein Ricin A Chain in Complex with the C-Terminal Peptide of the Ribosomal Stalk Protein P2

    PubMed Central

    Shi, Wei-Wei; Tang, Yun-Sang; Sze, See-Yuen; Zhu, Zhen-Ning; Wong, Kam-Bo; Shaw, Pang-Chui

    2016-01-01

    Ricin is a type 2 ribosome-inactivating protein (RIP), containing a catalytic A chain and a lectin-like B chain. It inhibits protein synthesis by depurinating the N-glycosidic bond at α-sarcin/ricin loop (SRL) of the 28S rRNA, which thereby prevents the binding of elongation factors to the GTPase activation center of the ribosome. Here, we present the 1.6 Å crystal structure of Ricin A chain (RTA) complexed to the C-terminal peptide of the ribosomal stalk protein P2, which plays a crucial role in specific recognition of elongation factors and recruitment of eukaryote-specific RIPs to the ribosomes. Our structure reveals that the C-terminal GFGLFD motif of P2 peptide is inserted into a hydrophobic pocket of RTA, while the interaction assays demonstrate the structurally untraced SDDDM motif of P2 peptide contributes to the interaction with RTA. This interaction mode of RTA and P protein is in contrast to that with trichosanthin (TCS), Shiga-toxin (Stx) and the active form of maize RIP (MOD), implying the flexibility of the P2 peptide-RIP interaction, for the latter to gain access to ribosome. PMID:27754366

  1. Lectin Receptor Kinases Participate in Protein-Protein Interactions to Mediate Plasma Membrane-Cell Wall Adhesions in Arabidopsis1

    PubMed Central

    Gouget, Anne; Senchou, Virginie; Govers, Francine; Sanson, Arnaud; Barre, Annick; Rougé, Pierre; Pont-Lezica, Rafael; Canut, Hervé

    2006-01-01

    Interactions between plant cell walls and plasma membranes are essential for cells to function properly, but the molecules that mediate the structural continuity between wall and membrane are unknown. Some of these interactions, which are visualized upon tissue plasmolysis in Arabidopsis (Arabidopsis thaliana), are disrupted by the RGD (arginine-glycine-aspartic acid) tripeptide sequence, a characteristic cell adhesion motif in mammals. In planta induced-O (IPI-O) is an RGD-containing protein from the plant pathogen Phytophthora infestans that can disrupt cell wall-plasma membrane adhesions through its RGD motif. To identify peptide sequences that specifically bind the RGD motif of the IPI-O protein and potentially play a role in receptor recognition, we screened a heptamer peptide library displayed in a filamentous phage and selected two peptides acting as inhibitors of the plasma membrane RGD-binding activity of Arabidopsis. Moreover, the two peptides also disrupted cell wall-plasma membrane adhesions. Sequence comparison of the RGD-binding peptides with the Arabidopsis proteome revealed 12 proteins containing amino acid sequences in their extracellular domains common with the two RGD-binding peptides. Eight belong to the receptor-like kinase family, four of which have a lectin-like extracellular domain. The lectin domain of one of these, At5g60300, recognized the RGD motif both in peptides and proteins. These results imply that lectin receptor kinases are involved in protein-protein interactions with RGD-containing proteins as potential ligands, and play a structural and signaling role at the plant cell surfaces. PMID:16361528

  2. Lectin receptor kinases participate in protein-protein interactions to mediate plasma membrane-cell wall adhesions in Arabidopsis.

    PubMed

    Gouget, Anne; Senchou, Virginie; Govers, Francine; Sanson, Arnaud; Barre, Annick; Rougé, Pierre; Pont-Lezica, Rafael; Canut, Hervé

    2006-01-01

    Interactions between plant cell walls and plasma membranes are essential for cells to function properly, but the molecules that mediate the structural continuity between wall and membrane are unknown. Some of these interactions, which are visualized upon tissue plasmolysis in Arabidopsis (Arabidopsis thaliana), are disrupted by the RGD (arginine-glycine-aspartic acid) tripeptide sequence, a characteristic cell adhesion motif in mammals. In planta induced-O (IPI-O) is an RGD-containing protein from the plant pathogen Phytophthora infestans that can disrupt cell wall-plasma membrane adhesions through its RGD motif. To identify peptide sequences that specifically bind the RGD motif of the IPI-O protein and potentially play a role in receptor recognition, we screened a heptamer peptide library displayed in a filamentous phage and selected two peptides acting as inhibitors of the plasma membrane RGD-binding activity of Arabidopsis. Moreover, the two peptides also disrupted cell wall-plasma membrane adhesions. Sequence comparison of the RGD-binding peptides with the Arabidopsis proteome revealed 12 proteins containing amino acid sequences in their extracellular domains common with the two RGD-binding peptides. Eight belong to the receptor-like kinase family, four of which have a lectin-like extracellular domain. The lectin domain of one of these, At5g60300, recognized the RGD motif both in peptides and proteins. These results imply that lectin receptor kinases are involved in protein-protein interactions with RGD-containing proteins as potential ligands, and play a structural and signaling role at the plant cell surfaces.

  3. Protein content and freezing avoidance properties of the subdermal extracellular matrix and serum of the Antarctic snailfish, Paraliparis devriesi.

    PubMed

    Jung, A; Johnson, P; Eastman, J T; Devries, A L

    1995-02-01

    The Antarctic snailfish, Paraliparis devriesi (Liparididae), occupies an epibenthic habitat at a depth of 500-650 m in the subzero waters of McMurdo Sound, Antarctica. This species has watery (97%) gelatinous subdermal extracellular matrix (SECM) comprising a mean of 33.8% of the body weight, the largest known proportion of any adult fish. The protein concentration of the SECM was found to be 6-7 mg ml(-1) (0.6-0.7% w/v). Separation of the polypeptides of the SECM by SDS-PAGE revealed 11 polypeptides ranging in relative molecular mass (Mr) from 67,000 to 13,000, with other unresolved polypeptides of less than 13,000. The isoelectric points of these proteins ranged from 4.85 to 8.05. Partial N-terminal amino acid sequence data were obtained for four of the major SECM polypeptides. The N-terminal amino acid sequences of three of these were not identical to or homologous with any other known sequences, whereas the N-terminal sequence of one polypeptide (Mr 51,000) was identical to partial sequence from the apolipoprotein A-I precursor of Atlantic salmon (Salmo salar). Although not isolated from either SECM or serum, melting point-freezing point behavior of body fluids suggest that Paraliparis possess modest amounts of a noncolligative antifreeze compound. Since relatively small amounts of antifreeze are present in the serum and even less in the SECM, freezing avoidance results from the combined effects of antifreeze and the elevated osmolality of body fluids. There are no special adaptations to prevent freezing in the superficially located high water content SECM.

  4. Assignment of the human pulmonary surfactant protein D gene (SFTP4) to 10q22-q23 close to the surfactant protein A gene cluster

    SciTech Connect

    Koelble, K.; Kaluz, S.; Reid, K.B.M. ); Mole, S.E. )

    1993-08-01

    Pulmonary surfactant consists of a complex mixture of phospholipids and several proteins essential to normal respiratory function. Two of the surfactant proteins, SP-A and SP-D, appear to have lectin-like activity relevant to the local phagocytic defense. Using polymerase chain reaction (PCR)-based somatic cell hybrid mapping, the human SP-D gene (SFTP4) was assigned to chromosome 10. A regional mapping panel was assembled and characterized using sequence tagged sites for five loci previously mapped to 10q. SFTP4, the SP-A gene (SFTP1), and the microsatellite D10S109 were placed in the interval 10q22-q23. Low-stringency PCR using the SFTP1 primer pair suggested the presence of at least two additional SP-A-related genes in the same region. With the locus for mannose-binding lectin (MBL) at 10q21, this may be indicative of this region's central role in the evolutionary history of carbohydrate-binding proteins containing collagen-like regions. 41 refs., 3 figs., 1 tab.

  5. Genetic Enhancement of an Anti-Freeze Protein for use as a Substitute for Ethylene Glycol for Aircraft Anti-icing

    DTIC Science & Technology

    2001-10-01

    in the solution (water) but not on its nature, and therefore freezing point depression is called a " colligative property ", denoting "depending on the collection". 2 ...programs costing over $8.2M. Additionally, an Air Force policy has been issued banning future purchase of ethylene glycol. Traditional Colligative

  6. Regulation of antifreeze protein messenger RNA and a female-specific messenger RNA in winter flounder: Evidence for temperature and photoperiod effects

    SciTech Connect

    Price, J.L.

    1988-01-01

    Northern blot hybridizations showed that mRNA{sub f} is female-specific, and that both mRNAs are liver-specific and expressed at high levels only during the fall and winter in the wild. When warm-acclimated flounder (18C) were acclimated over 2 days to low temperature (4 C) and short photoperiod in July and October, high levels of AF mRNA accumulated within 4-10 days. Low levels were found in fish at high temperature at any time, on long photoperiod after acclimation to low temperature in July, or continuously at 4 C from March until September. A temperature increase in January resulted in a dramatic decrease in the levels of AF mRNA within 5 days. Beta actin mRNA, mRNA{sub f} and most mRNAs translated in a reticulocyte lysate did not change in abundance after temperature increases or decreases. Synthesis of AF mRNA in liver slices was investigated by Northern blot hybridization and hybridization of {sup 32}P-labeled RNA to AF cDNA. Liver slices from cold-acclimated January fish expressed high levels of AF mRNA initially at 4 C and lower levels at 18 C. AF mRNA expression was turned off in tissue from warm-acclimated fish but was progressively induced after long 4 C incubations in vitro in livers from fall fish but not from spring fish. Temperature and photoperiod act either as zeitgebers, which set the phase of an endogenous rhythm, or as more direct signals that stimulate a transient accumulation of AF mRNA.

  7. Effects of a type I antifreeze protein (AFP) on the melting of frozen AFP and AFP+solute aqueous solutions studied by NMR microimaging experiment.

    PubMed

    Ba, Yong; Mao, Yougang; Galdino, Luiz; Günsen, Zorigoo

    2013-01-01

    The effects of a type I AFP on the bulk melting of frozen AFP solutions and frozen AFP+solute solutions were studied through an NMR microimaging experiment. The solutes studied include sodium chloride and glucose and the amino acids alanine, threonine, arginine, and aspartic acid. We found that the AFP is able to induce the bulk melting of the frozen AFP solutions at temperatures lower than 0 °C and can also keep the ice melted at higher temperatures in the AFP+solute solutions than those in the corresponding solute solutions. The latter shows that the ice phases were in super-heated states in the frozen AFP+solute solutions. We have tried to understand the first experimental phenomenon via the recent theoretical prediction that type I AFP can induce the local melting of ice upon adsorption to ice surfaces. The latter experimental phenomenon was explained with the hypothesis that the adsorption of AFP to ice surfaces introduces a less hydrophilic water-AFP-ice interfacial region, which repels the ionic/hydrophilic solutes. Thus, this interfacial region formed an intermediate chemical potential layer between the water phase and the ice phase, which prevented the transfer of water from the ice phase to the water phase. We have also attempted to understand the significance of the observed melting phenomena to the survival of organisms that express AFPs over cold winters.

  8. Probing protein-sugar interactions.

    PubMed

    Ebel, C; Eisenberg, H; Ghirlando, R

    2000-01-01

    We have investigated the partial specific volumes (2) (ml/g), hydration, and cosolvent interactions of rabbit muscle aldolase by equilibrium sedimentation in the analytical ultracentrifuge and by direct density increment (partial differential/partial differentialc(2))(mu) measurements over a range of sugar concentrations and temperature. In a series of sugars increasing in size, glucose, sucrose, raffinose, and alpha-cyclodextrin, (partial differential/ partial differentialc(2))(mu) decreases linearly with the solvent density rho(0). These sugar cosolvents do not interact with the protein; however, the interaction parameter B(1) (g water/g protein) mildly increases with increasing sugar size. The experimental B(1) values are smaller than values calculated by excluded volume (rolling ball) considerations. B(1) relates to hydration in this and in other instances studied. It decreases with increasing temperature, leading to an increase in (2) due to reduced water of hydration electrostriction. The density increments (partial differential/ partial differentialc(2))(mu), however, decrease in concave up form in the case of glycerol and in concave down form for trehalose, leading to more complex behavior in the case of carbohydrates playing a biological role as osmolytes and antifreeze agents. A critical discussion, based on the thermodynamics of multicomponent solutions, is presented.

  9. Insecticidal activity of wheat Hessian fly responsive proteins HFR-1 and HFR-3 towards a non-target wheat pest, cereal aphid (Sitobion avenae F.).

    PubMed

    Pyati, Prashant; Chellamuthu, Anitha; Gatehouse, Angharad M R; Fitches, Elaine; Gatehouse, John A

    2012-07-01

    The interaction between Hessian fly (Mayetiola destructor) and wheat (Triticum aestivum) involves a gene-for-gene resistance mechanism. The incompatible interaction leading to resistance involves up-regulation of several Hfr (Hessian fly responsive) genes encoding proteins with potential insecticidal activity. The encoded proteins HFR-1, HFR-2 and HFR-3 all possess lectin-like domains. HFR-1 and HFR-3 were produced as recombinant proteins using Escherichia coli and Pichia pastoris, respectively as expression hosts. Purified recombinant proteins were assayed for insecticidal effects towards cereal aphid (Sitobion avenae), an insect to which wheat shows only tolerance. Both HFR-1 and HFR-3 were found to be insecticidal towards S. avenae when fed in artificial diet. Although HFR-3 has sequence similarity and similar chitin-binding activity to wheat germ agglutinin (WGA), the latter protein was almost non-toxic to S. avenae. HFR-3 binds strongly to aphid midguts after ingestion, whereas WGA binds but does not persist over a feed-chase period. Quantitative PCR showed that Hfr-3 mRNA does not increase in level after cereal aphid infestation. The results suggest that the lack of effective resistance to cereal aphid in wheat is not due to an absence of genes encoding suitable insecticidal proteins, but results from a failure to up-regulate gene expression in response to aphid attack.

  10. Ice-binding proteins: a remarkable diversity of structures for stopping and starting ice growth.

    PubMed

    Davies, Peter L

    2014-11-01

    Antifreeze proteins (AFPs) were discovered in marine fishes that need protection from freezing. These ice-binding proteins (IBPs) are widespread across biological kingdoms, and their functions include freeze tolerance and ice adhesion. Consistent with recent independent evolution, AFPs have remarkably diverse folds that rely heavily on hydrogen- and disulfide-bonding. AFP ice-binding sites are typically flat, extensive, relatively hydrophobic, and are thought to organize water into an ice-like arrangement that merges and freezes with the quasi-liquid layer next to the ice lattice. In this article, the roles, properties, and structure-function interactions of IBPs are reviewed, and their relationship to ice nucleation proteins, which promote freezing at high subzero temperatures, is explored.

  11. Adaptive evolution of the venom-targeted vWF protein in opossums that eat pitvipers.

    PubMed

    Jansa, Sharon A; Voss, Robert S

    2011-01-01

    The rapid evolution of venom toxin genes is often explained as the result of a biochemical arms race between venomous animals and their prey. However, it is not clear that an arms race analogy is appropriate in this context because there is no published evidence for rapid evolution in genes that might confer toxin resistance among routinely envenomed species. Here we report such evidence from an unusual predator-prey relationship between opossums (Marsupialia: Didelphidae) and pitvipers (Serpentes: Crotalinae). In particular, we found high ratios of replacement to silent substitutions in the gene encoding von Willebrand Factor (vWF), a venom-targeted hemostatic blood protein, in a clade of opossums known to eat pitvipers and to be resistant to their hemorrhagic venom. Observed amino-acid substitutions in venom-resistant opossums include changes in net charge and hydrophobicity that are hypothesized to weaken the bond between vWF and one of its toxic snake-venom ligands, the C-type lectin-like protein botrocetin. Our results provide the first example of rapid adaptive evolution in any venom-targeted molecule, and they support the notion that an evolutionary arms race might be driving the rapid evolution of snake venoms. However, in the arms race implied by our results, venomous snakes are prey, and their venom has a correspondingly defensive function in addition to its usual trophic role.

  12. Characterization of the ice-binding protein from Arctic yeast Leucosporidium sp. AY30.

    PubMed

    Park, Kyoung Sun; Do, Hackwon; Lee, Jun Hyuck; Park, Seung Il; Kim, Eun jung; Kim, Soon-Jong; Kang, Sung-Ho; Kim, Hak Jun

    2012-06-01

    Previously, we reported the ice-binding protein (LeIBP) from the Arctic yeast Leucosporidium sp. AY30. In this study we provide physicochemical characterization of this IBP, which belongs to a class of IBPs that exhibited no significant similarity in primary structure to other known antifreeze proteins (AFPs). We compared native, glycosylated and non-glycosylated recombinant LeIBPs. Interestingly, size-exclusion chromatography and analytical ultracentrifugation revealed that LeIBP self-associates with a reversible dimer with K(d) values in the range 3.45-7.24×10(-6) M. Circular dichroism (CD) spectra showed that LeIBP, glycosylated or non-glycosylated, is predominantly composed of β-strand secondary structural elements (54.6%), similar to other β-helical antifreeze proteins (AFPs). In thermal hysteresis (TH) activity measurements, native LeIBP was twice more active (0.87 °C at 15 mg/mL) than that of the recombinant IBPs (0.43-0.42 °C at 10.8 mg/mL). This discrepancy is probably due to uncharacterized enhancing factors carried over during ice affinity purification, because glycosylated and non-glycosylated recombinant proteins displayed similarly low activity. Ice recrystallization inhibition (RI) activities of the native and recombinant LeIBPs were comparable. Measurements of CD, TH activity, and RI showed that glycosylation does not cause structural changes and is not required for function. An ice-etching experiment using green fluorescent protein-tagged IBP revealed that LeIBP binds, just as hyperactive AFPs, to both basal and pyramidal prism planes of the ice crystal. Taken together, our results indicate that LeIBP, structurally similar to hyperactive AFPs, is moderately active and that a reversible dimer has no effect on its activity.

  13. Modulating Endoplasmic Reticulum-Golgi Cargo Receptors for Improving Secretion of Carrier-Fused Heterologous Proteins in the Filamentous Fungus Aspergillus oryzae

    PubMed Central

    Hoang, Huy-Dung; Maruyama, Jun-ichi

    2014-01-01

    Filamentous fungi are excellent hosts for industrial protein production due to their superior secretory capacity; however, the yield of heterologous eukaryotic proteins is generally lower than that of fungal or endogenous proteins. Although activating protein folding machinery in the endoplasmic reticulum (ER) improves the yield, the importance of intracellular transport machinery for heterologous protein secretion is poorly understood. Here, using Aspergillus oryzae as a model filamentous fungus, we studied the involvement of two putative lectin-like cargo receptors, A. oryzae Vip36 (AoVip36) and AoEmp47, in the secretion of heterologous proteins expressed in fusion with the endogenous enzyme α-amylase as the carrier. Fluorescence microscopy revealed that mDsRed-tagged AoVip36 localized in the Golgi compartment, whereas AoEmp47 showed localization in both the ER and the Golgi compartment. Deletion of AoVip36 and AoEmp47 improved heterologous protein secretion, but only AoVip36 deletion had a negative effect on the secretion of α-amylase. Analysis of ER-enriched cell fractions revealed that AoVip36 and AoEmp47 were involved in the retention of heterologous proteins in the ER. However, the overexpression of each cargo receptor had a different effect on heterologous protein secretion: AoVip36 enhanced the secretion, whereas AoEmp47 promoted the intracellular retention. Taken together, our data suggest that AoVip36 and AoEmp47 hinder the secretion of heterologous proteins by promoting their retention in the ER but that AoVip36 also promotes the secretion of heterologous proteins. Moreover, we found that genetic deletion of these putative ER-Golgi cargo receptors significantly improves heterologous protein production. The present study is the first to propose that ER-Golgi transport is a bottleneck for heterologous protein production in filamentous fungi. PMID:25362068

  14. Water-mediated influence of a crowded environment on internal vibrations of a protein molecule.

    PubMed

    Kuffel, Anna; Zielkiewicz, Jan

    2016-02-14

    The influence of crowding on the protein inner dynamics is examined by putting a single protein molecule close to one or two neighboring protein molecules. The presence of additional molecules influences the amplitudes of protein fluctuations. Also, a weak dynamical coupling of collective velocities of surface atoms of proteins separated by a layer of water is detected. The possible mechanisms of these phenomena are described. The cross-correlation function of the collective velocities of surface atoms of two proteins was decomposed into the Fourier series. The amplitude spectrum displays a peak at low frequencies. Also, the results of principal component analysis suggest that the close presence of an additional protein molecule influences the high-amplitude, low-frequency modes in the most prominent way. This part of the spectrum covers biologically important protein motions. The neighbor-induced changes in the inner dynamics of the protein may be connected with the changes in the velocity power spectrum of interfacial water. The additional protein molecule changes the properties of solvation water and in this way it can influence the dynamics of the second protein. It is suggested that this phenomenon may be described, at first approximation, by a damped oscillator driven by an external random force. This model was successfully applied to conformationally rigid Choristoneura fumiferana antifreeze protein molecules.

  15. Why are hyperactive ice-binding-proteins so active?

    NASA Astrophysics Data System (ADS)

    Braslavsky, Ido; Celik, Yeliz; Pertaya, Natalya; Eun Choi, Young; Bar, Maya; Davies, Peter L.

    2008-03-01

    Ice binding proteins (IBPs), also called `antifreeze proteins' or `ice structuring proteins', are a class of proteins that protect organisms from freezing injury. These proteins have many applications in medicine and agriculture, and as a platform for future biotechnology applications. One of the interesting questions in this field focuses on the hyperactivity of some IBPs. Ice binding proteins can be classified in two groups: moderate ones that can depress the freezing point up to ˜1.0 ^oC and hyperactive ones that can depress the freezing point several-fold further even at lower concentrations. It has been suggested that the hyperactivity of IBPs stem from the fact that they block growth out of specific ice surfaces, more specifically the basal planes of ice. Here we show experimental results based on fluorescence microscopy, highlighting the differences between moderate IBPs and hyperactive IBPs. These include direct evidence for basal plane affinity of hyperactive IBPs, the effects of IBPs on growth-melt behavior of ice and the dynamics of their interaction with ice.

  16. Membrane-bound heat shock proteins facilitate the uptake of dying cells and cross-presentation of cellular antigen.

    PubMed

    Zhu, Haiyan; Fang, Xiaoyun; Zhang, Dongmei; Wu, Weicheng; Shao, Miaomiao; Wang, Lan; Gu, Jianxin

    2016-01-01

    Heat shock proteins (HSPs) were originally identified as stress-responsive proteins and serve as molecular chaperones in different intracellular compartments. Translocation of HSPs to the cell surface and release of HSPs into the extracellular space have been observed during the apoptotic process and in response to a variety of cellular stress. Here, we report that UV irradiation and cisplatin treatment rapidly induce the expression of membrane-bound Hsp60, Hsp70, and Hsp90 upstream the phosphatidylserine exposure. Membrane-bound Hsp60, Hsp70 and Hsp90 could promote the release of IL-6 and IL-1β as well as DC maturation by the evaluation of CD80 and CD86 expression. On the other hand, Hsp60, Hsp70 and Hsp90 on cells could facilitate the uptake of dying cells by bone marrow-derived dendritic cells. Lectin-like oxidized LDL receptor-1 (LOX-1), as a common receptor for Hsp60, Hsp70, and Hsp90, is response for their recognition and mediates the uptake of dying cells. Furthermore, membrane-bound Hsp60, Hsp70 and Hsp90 could promote the cross-presentation of OVA antigen from E.G7 cells and inhibition of the uptake of dying cells by LOX-1 decreases the cross-presentation of cellular antigen. Therefore, the rapid exposure of HSPs on dying cells at the early stage allows for the recognition by and confers an activation signal to the immune system.

  17. Flavobacterium columnare: Chemotaxis and adhesion to channel catfish mucus is mediated by lectin-like capsular substances

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Flavobacterium columnare is an important Gram-negative pathogen of fresh water fish that may cause chronic skin lesions and severe mortality. Isolates of F. columnare belong to either the virulent genomovar II or the less virulent genomovar I. Chemotaxis and adhesion assays were conducted in vitro...

  18. Modeling Pseudomonas syringae ice-nucleation protein as a beta-helical protein.

    PubMed Central

    Graether, S P; Jia, Z

    2001-01-01

    Antifreeze proteins (AFPs) inhibit the growth of ice, whereas ice-nucleation proteins (INPs) promote its formation. Although the structures of several AFPs are known, the structure of INP has been modeled thus far because of the difficulty in determining membrane protein structures. Here, we present a novel model of an INP structure from Pseudomonas syringae based on comparison with two newly determined insect AFP structures. The results suggest that both this class of AFPs and INPs may have a similar beta-helical fold and that they could interact with water through the repetitive TXT motif. By theoretical arguments, we show that the distinguishing feature between an ice inhibitor and an ice nucleator lies in the size of the ice-interacting surface. For INPs, the larger surface area acts as a template that is larger than the critical ice embryo surface area required for growth. In contrast, AFPs are small enough so that they bind to ice and inhibit further growth without acting as a nucleator. PMID:11222281

  19. Identification of the N-acetylneuraminyllactose-specific laminin-binding protein of Helicobacter pylori.

    PubMed Central

    Valkonen, K H; Wadström, T; Moran, A P

    1997-01-01

    The interaction of the gastroduodenal pathogen Helicobacter pylori with the glycoprotein laminin was investigated. Binding of 125I-radiolabelled laminin in a liquid-phase assay by both hemagglutinating and poorly hemagglutinating strains was rapid, saturable, specific, partially reversible, of high affinity, and insensitive to pH. Inhibition of laminin binding by fetuin, but not asialofetuin, and reduced bacterial binding to periodate- or sialidase-treated laminin indicated that glycosylation, particularly sialylation, was important for laminin binding by H. pylori. Inhibition experiments with monosaccharides, disaccharides, and trisaccharides showed that the strains bound to a region spanning a trisaccharide. In particular, inhibition and displacement studies showed that binding to the trisaccharide N-acetylneuraminyl-alpha(2-3)-lactose [NeuAc(2-3)Lac] was preferential to that to the NeuAc(2-6)Lac isomer. Complete inhibition of laminin binding by both hemagglutinating and poorly hemagglutinating strains was achieved only when isolated lipopolysaccharide (LPS) was used as an inhibitor in combination with heat or protease treatment of H. pylori cells, thereby confirming the involvement of both LPS and a protein adhesin in laminin binding. Further inhibition experiments indicated that the protein receptor, rather than LPS, on H. pylori bound NeuAc(2-3)Lac. By using a Western blotting procedure, a 25-kDa outer membrane protein was identified as mediating laminin binding by both hemagglutinating and poorly hemagglutinating H. pylori strains. The specificity of binding was confirmed by complete inhibition of laminin binding by the 25-kDa protein with NeuAc(2-3)Lac. The data collectively suggest that a 25-kDa outer membrane protein acts in a lectin-like manner with LPS to mediate attachment of H. pylori to laminin. PMID:9038297

  20. On the ability of molecular dynamics simulation and continuum electrostatics to treat interfacial water molecules in protein-protein complexes

    PubMed Central

    Copie, Guillaume; Cleri, Fabrizio; Blossey, Ralf; Lensink, Marc F.

    2016-01-01

    Interfacial waters are increasingly appreciated as playing a key role in protein-protein interactions. We report on a study of the prediction of interfacial water positions by both Molecular Dynamics and explicit solvent-continuum electrostatics based on the Dipolar Poisson-Boltzmann Langevin (DPBL) model, for three test cases: (i) the barnase/barstar complex (ii) the complex between the DNase domain of colicin E2 and its cognate Im2 immunity protein and (iii) the highly unusual anti-freeze protein Maxi which contains a large number of waters in its interior. We characterize the waters at the interface and in the core of the Maxi protein by the statistics of correctly predicted positions with respect to crystallographic water positions in the PDB files as well as the dynamic measures of diffusion constants and position lifetimes. Our approach provides a methodology for the evaluation of predicted interfacial water positions through an investigation of water-mediated inter-chain contacts. While our results show satisfactory behaviour for molecular dynamics simulation, they also highlight the need for improvement of continuum methods. PMID:27905545

  1. Crystallization and preliminary X-ray analysis of coagulation factor IX-binding protein from habu snake venom at pH 6.5 and 4.6

    SciTech Connect

    Suzuki, Nobuhiro; Shikamoto, Yasuo; Fujimoto, Zui; Morita, Takashi; Mizuno, Hiroshi

    2005-01-01

    Crystals of habu coagulation factor IX-binding protein have been obtained at pH 6.5 and 4.6 and characterized by X-ray diffraction. Coagulation factor IX-binding protein isolated from Trimeresurus flavoviridis (IX-bp) is a C-type lectin-like protein. It is an anticoagulant protein consisting of homologous subunits A and B. The subunits both contain a Ca{sup 2+}-binding site with differing affinity (K{sub d} values of 14 and 130 µM at pH 7.5). These binding characteristics are pH-dependent; under acidic conditions, the affinity of the low-affinity site was reduced considerably. In order to identify which site has high affinity and also to investigate the Ca{sup 2+}-releasing mechanism, IX-bp was crystallized at pH 6.5 and 4.6. The crystals at pH 6.5 and 4.6 diffracted to 1.72 and 2.29 Å resolution, respectively; the former crystals belong to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 60.7, b = 63.5, c = 66.9 Å, β = 117.0°, while the latter belong to the monoclinic space group C2, with a = 134.1, b = 37.8, c = 55.8 Å, β = 110.4°.

  2. Convergent Synthesis of Homogeneous Glc1Man9GlcNAc2-Protein and Derivatives as Ligands of Molecular Chaperones in Protein Quality Control

    PubMed Central

    Amin, Mohammed N.; Huang, Wei; Mizanur, Rahman M.

    2011-01-01

    A detailed understanding of the molecular mechanism of chaperone-assisted protein quality control is often hampered by the lack of well-defined homogeneous glycoprotein probes. We describe here a highly convergent chemoenzymatic synthesis of the monoglucosylated glycoforms of bovine ribonuclease (RNase) as specific ligands of lectin-like chaperones calnexin (CNX) and calreticulin (CRT) that are known to recognize the monoglucosylated high-mannose oligosaccharide component of glycoproteins in protein folding. The synthesis of a selectively modified glycoform Gal1Glc1Man9GlcNAc2-RNase was accomplished by chemical synthesis of a large N-glycan oxazoline and its subsequent enzymatic ligation to GlcNAc-RNase under the catalysis of a glycosynthase. Selective removal of the terminal galactose by a β-galactosidase gave the Glc1Man9GlcNAc2-RNase glycoform in excellent yield. CD spectroscopic analysis and RNA-hydrolyzing assay indicated that the synthetic RNase glycoforms maintained essentially the same global conformations and were fully active as the natural bovine ribonuclease B. SPR binding studies revealed that the Glc1Man9GlcNAc2-RNase had high affinity to lectin CRT, while the synthetic Man9GlcNAc2-RNase glycoform and natural RNase B did not show CRT-binding activity. These results confirmed the essential role of the glucose moiety in the chaperone molecular recognition. Interestingly, the galactose-masked glycoform Gal1Glc1Man9GlcNAc2-RNase also showed significant affinity to lectin CRT, suggesting that a galactose β-1,4-linked to the key glucose moiety does not significantly block the lectin binding. These synthetic homogeneous glycoprotein probes should be valuable for a detailed mechanistic study on how molecular chaperones work in concert to distinguish between mis-folded and folded glycoproteins in the protein quality control cycle. PMID:21819116

  3. Renal mass reduction results in accumulation of lipids and dysregulation of lipid regulatory proteins in the remnant kidney.

    PubMed

    Kim, Hyun Ju; Moradi, Hamid; Yuan, Jun; Norris, Keith; Vaziri, Nosratola D

    2009-06-01

    A significant reduction of renal mass results in proteinuria, glomerulosclerosis, and tubulointerstitial injury, culminating in end-stage chronic renal failure (CRF). The accumulation of lipids in the kidney can cause renal disease. Uptake of oxidized lipoproteins via scavenger receptors, reabsorption of filtered protein-bound lipids via the megalin-cubilin complex, and increased glucose load per nephron can promote lipid accumulation in glomerular, tubular, and interstitial cells in CRF. Cellular lipid homeostasis is regulated by lipid influx, synthesis, catabolism, and efflux. We examined lipid-regulatory factors in the remnant kidney of rats 11 wk after nephrectomy (CRF) or sham operation. CRF resulted in azotemia, proteinuria, lipid accumulation in the kidney, upregulation of megalin, cubilin, mediators of lipid influx (scavenger receptor class A and lectin-like oxidized receptor-1), lipid efflux (liver X receptor alpha/beta and ATP-binding cassette transporter), and fatty acid biosynthesis (carbohydrate-response element binding protein, fatty acid synthase, and acetyl-CoA carboxylase). However, factors involved in cholesterol biosynthesis (sterol regulatory element binding protein, 3-hydroxy-3-methylglutaryl coenzyme A reductase, SCAP, Insig-1, and Insig-2) and fatty acid oxidation (peroxisome proliferator-activated receptor, acyl-CoA oxidase, and liver-type fatty acid binding protein) were reduced in the remnant kidney. Thus CRF results in heavy lipid accumulation in the remnant kidney, which is mediated by upregulation of pathways involved in tubular reabsorption of filtered protein-bound lipids, influx of oxidized lipoproteins and synthesis of fatty acids, and downregulation of pathways involved in fatty acid catabolism.

  4. Natural Compounds Interacting with Nicotinic Acetylcholine Receptors: From Low-Molecular Weight Ones to Peptides and Proteins

    PubMed Central

    Kudryavtsev, Denis; Shelukhina, Irina; Vulfius, Catherine; Makarieva, Tatyana; Stonik, Valentin; Zhmak, Maxim; Ivanov, Igor; Kasheverov, Igor; Utkin, Yuri; Tsetlin, Victor

    2015-01-01

    Nicotinic acetylcholine receptors (nAChRs) fulfill a variety of functions making identification and analysis of nAChR subtypes a challenging task. Traditional instruments for nAChR research are d-tubocurarine, snake venom protein α-bungarotoxin (α-Bgt), and α-conotoxins, neurotoxic peptides from Conus snails. Various new compounds of different structural classes also interacting with nAChRs have been recently identified. Among the low-molecular weight compounds are alkaloids pibocin, varacin and makaluvamines C and G. 6-Bromohypaphorine from the mollusk Hermissenda crassicornis does not bind to Torpedo nAChR but behaves as an agonist on human α7 nAChR. To get more selective α-conotoxins, computer modeling of their complexes with acetylcholine-binding proteins and distinct nAChRs was used. Several novel three-finger neurotoxins targeting nAChRs were described and α-Bgt inhibition of GABA-A receptors was discovered. Information on the mechanisms of nAChR interactions with the three-finger proteins of the Ly6 family was found. Snake venom phospholipases A2 were recently found to inhibit different nAChR subtypes. Blocking of nAChRs in Lymnaea stagnalis neurons was shown for venom C-type lectin-like proteins, appearing to be the largest molecules capable to interact with the receptor. A huge nAChR molecule sensible to conformational rearrangements accommodates diverse binding sites recognizable by structurally very different compounds. PMID:26008231

  5. Flies expand the repertoire of protein structures that bind ice

    PubMed Central

    Basu, Koli; Graham, Laurie A.; Campbell, Robert L.; Davies, Peter L.

    2015-01-01

    An antifreeze protein (AFP) with no known homologs has been identified in Lake Ontario midges (Chironomidae). The midge AFP is expressed as a family of isoforms at low levels in adults, which emerge from fresh water in spring before the threat of freezing temperatures has passed. The 9.1-kDa major isoform derived from a preproprotein precursor is glycosylated and has a 10-residue tandem repeating sequence xxCxGxYCxG, with regularly spaced cysteines, glycines, and tyrosines comprising one-half its 79 residues. Modeling and molecular dynamics predict a tightly wound left-handed solenoid fold in which the cysteines form a disulfide core to brace each of the eight 10-residue coils. The solenoid is reinforced by intrachain hydrogen bonds, side-chain salt bridges, and a row of seven stacked tyrosines on the hydrophobic side that forms the putative ice-binding site. A disulfide core is also a feature of the similar-sized beetle AFP that is a β-helix with seven 12-residue coils and a comparable circular dichroism spectrum. The midge and beetle AFPs are not homologous and their ice-binding sites are radically different, with the latter comprising two parallel arrays of outward-pointing threonines. However, their structural similarities is an amazing example of convergent evolution in different orders of insects to cope with change to a colder climate and provide confirmation about the physical features needed for a protein to bind ice. PMID:25561557

  6. Genomic Analysis of Storage Protein Deficiency in Genetically Related Lines of Common Bean (Phaseolus vulgaris)

    PubMed Central

    Pandurangan, Sudhakar; Diapari, Marwan; Yin, Fuqiang; Munholland, Seth; Perry, Gregory E.; Chapman, B. Patrick; Huang, Shangzhi; Sparvoli, Francesca; Bollini, Roberto; Crosby, William L.; Pauls, Karl P.; Marsolais, Frédéric

    2016-01-01

    A series of genetically related lines of common bean (Phaseolus vulgaris L.) integrate a progressive deficiency in major storage proteins, the 7S globulin phaseolin and lectins. SARC1 integrates a lectin-like protein, arcelin-1 from a wild common bean accession. SMARC1N-PN1 is deficient in major lectins, including erythroagglutinating phytohemagglutinin (PHA-E) but not α-amylase inhibitor, and incorporates also a deficiency in phaseolin. SMARC1-PN1 is intermediate and shares the phaseolin deficiency. Sanilac is the parental background. To understand the genomic basis for variations in protein profiles previously determined by proteomics, the genotypes were submitted to short-fragment genome sequencing using an Illumina HiSeq 2000/2500 platform. Reads were aligned to reference sequences and subjected to de novo assembly. The results of the analyses identified polymorphisms responsible for the lack of specific storage proteins, as well as those associated with large differences in storage protein expression. SMARC1N-PN1 lacks the lectin genes pha-E and lec4-B17, and has the pseudogene pdlec1 in place of the functional pha-L gene. While the α-phaseolin gene appears absent, an approximately 20-fold decrease in β-phaseolin accumulation is associated with a single nucleotide polymorphism converting a G-box to an ACGT motif in the proximal promoter. Among residual lectins compensating for storage protein deficiency, mannose lectin FRIL and α-amylase inhibitor 1 genes are uniquely present in SMARC1N-PN1. An approximately 50-fold increase in α-amylase inhibitor like protein accumulation is associated with multiple polymorphisms introducing up to eight potential positive cis-regulatory elements in the proximal promoter specific to SMARC1N-PN1. An approximately 7-fold increase in accumulation of 11S globulin legumin is not associated with variation in proximal promoter sequence, suggesting that the identity of individual proteins involved in proteome rebalancing might

  7. Annealing condition influences thermal hysteresis of fungal type ice-binding proteins.

    PubMed

    Xiao, Nan; Hanada, Yuichi; Seki, Haruhiko; Kondo, Hidemasa; Tsuda, Sakae; Hoshino, Tamotsu

    2014-02-01

    The Antarctic sea ice diatom Navicular glaciei produced ice-binding protein (NagIBP) that is similar to the antifreeze protein (TisAFP) from snow mold Typhula ishikariensis. In the thermal hysteresis range of NagIBP, ice growth was completely inhibited. At the freezing point, the ice grew in a burst to 6 direction perdicular to the c-axis of ice crystal. This burst pattern is similar to TisAFP and other hyperactive AFPs. The thermal hysteresis of NagIBP and TisAFP could be increased by decreasing a cooling rate to allow more time for the proteins to bind ice. This suggests the possible second binding of proteins occurs on the ice surface, which might increase the hysteresises to a sufficient level to prevent freezing of the brine pockets which habitat of N. glaciei. The secondary ice binding was described as that after AFP molecules bind onto the flat ice plane irreversibly, which was based on adsorption-inhibition mechanism model at the ice-water interface, convex ice front was formed and overgrew during normal TH measurement (no annealing) until uncontrolled growth at the nonequilibrium freezing point. The results suggested that NagIBP is a hyperactive AFP that is expressed for freezing avoidance.

  8. Common protein sequence signatures associate with Sclerotinia borealis lifestyle and secretion in fungal pathogens of the Sclerotiniaceae

    PubMed Central

    Badet, Thomas; Peyraud, Rémi; Raffaele, Sylvain

    2015-01-01

    Fungal plant pathogens produce secreted proteins adapted to function outside fungal cells to facilitate colonization of their hosts. In many cases such as for fungi from the Sclerotiniaceae family the repertoire and function of secreted proteins remains elusive. In the Sclerotiniaceae, whereas Sclerotinia sclerotiorum and Botrytis cinerea are cosmopolitan broad host-range plant pathogens, Sclerotinia borealis has a psychrophilic lifestyle with a low optimal growth temperature, a narrow host range and geographic distribution. To spread successfully, S. borealis must synthesize proteins adapted to function in its specific environment. The search for signatures of adaptation to S. borealis lifestyle may therefore help revealing proteins critical for colonization of the environment by Sclerotiniaceae fungi. Here, we analyzed amino acids usage and intrinsic protein disorder in alignments of groups of orthologous proteins from the three Sclerotiniaceae species. We found that enrichment in Thr, depletion in Glu and Lys, and low disorder frequency in hot loops are significantly associated with S. borealis proteins. We designed an index to report bias in these properties and found that high index proteins were enriched among secreted proteins in the three Sclerotiniaceae fungi. High index proteins were also enriched in function associated with plant colonization in S. borealis, and in in planta-induced genes in S. sclerotiorum. We highlight a novel putative antifreeze protein and a novel putative lytic polysaccharide monooxygenase identified through our pipeline as candidate proteins involved in colonization of the environment. Our findings suggest that similar protein signatures associate with S. borealis lifestyle and with secretion in the Sclerotiniaceae. These signatures may be useful for identifying proteins of interest as targets for the management of plant diseases. PMID:26442085

  9. Properties and biotechnological applications of ice-binding proteins in bacteria.

    PubMed

    Cid, Fernanda P; Rilling, Joaquín I; Graether, Steffen P; Bravo, Leon A; Mora, María de La Luz; Jorquera, Milko A

    2016-06-01

    Ice-binding proteins (IBPs), such as antifreeze proteins (AFPs) and ice-nucleating proteins (INPs), have been described in diverse cold-adapted organisms, and their potential applications in biotechnology have been recognized in various fields. Currently, both IBPs are being applied to biotechnological processes, primarily in medicine and the food industry. However, our knowledge regarding the diversity of bacterial IBPs is limited; few studies have purified and characterized AFPs and INPs from bacteria. Phenotypically verified IBPs have been described in members belonging to Gammaproteobacteria, Actinobacteria and Flavobacteriia classes, whereas putative IBPs have been found in Gammaproteobacteria, Alphaproteobacteria and Bacilli classes. Thus, the main goal of this minireview is to summarize the current information on bacterial IBPs and their application in biotechnology, emphasizing the potential application in less explored fields such as agriculture. Investigations have suggested the use of INP-producing bacteria antagonists and AFPs-producing bacteria (or their AFPs) as a very attractive strategy to prevent frost damages in crops. UniProt database analyses of reported IBPs (phenotypically verified) and putative IBPs also show the limited information available on bacterial IBPs and indicate that major studies are required.

  10. Biophysical characterization of soluble Pseudomonas syringae ice nucleation protein InaZ fragments.

    PubMed

    Han, Yu Jin; Song, HyoJin; Lee, Chang Woo; Ly, Nguyễn Hoàng; Joo, Sang-Woo; Lee, Jun Hyuck; Kim, Soon-Jong; Park, SangYoun

    2017-01-01

    Ice nucleation protein (INP) with its functional domain consisting of multiple 48-residue repeat units effectively induces super-cooled water into ice. Circular dichroism and infrared deconvolution analyses on a soluble 240-residue fragment of Pseudomonas syringae InaZ (InaZ240) containing five 48-residue repeat units indicated that it is mostly composed of β-sheet and random coil. Analytical ultracentrifugation suggested that InaZ240 behaves as a monomer of an elongated ellipsoid. However, InaZ240 showed only minimum ice binding compared to anti-freeze proteins. Other P. syringae InaZ proteins with more 48-residue repeat units were made, in which the largest soluble fragment obtainable was an InaZ with twelve 48-residue repeat units. Size-exclusion chromatography analyses further suggested that the overall shape of the expressed InaZ fragments is pH-dependent, which becomes compact as the numbers of 48-residue repeat unit increase.

  11. A Secreted Protein with Plant-Specific Cysteine-Rich Motif Functions as a Mannose-Binding Lectin That Exhibits Antifungal Activity1[W

    PubMed Central

    Miyakawa, Takuya; Hatano, Ken-ichi; Miyauchi, Yumiko; Suwa, You-ichi; Sawano, Yoriko; Tanokura, Masaru

    2014-01-01

    Plants have a variety of mechanisms for defending against plant pathogens and tolerating environmental stresses such as drought and high salinity. Ginkbilobin2 (Gnk2) is a seed storage protein in gymnosperm that possesses antifungal activity and a plant-specific cysteine-rich motif (domain of unknown function26 [DUF26]). The Gnk2-homologous sequence is also observed in an extracellular region of cysteine-rich repeat receptor-like kinases that function in response to biotic and abiotic stresses. Here, we report the lectin-like molecular function of Gnk2 and the structural basis of its monosaccharide recognition. Nuclear magnetic resonance experiments showed that mannan was the only yeast (Saccharomyces cerevisiae) cell wall polysaccharide that interacted with Gnk2. Gnk2 also interacted with mannose, a building block of mannan, with a specificity that was similar to those of mannose-binding legume lectins, by strictly recognizing the configuration of the hydroxy group at the C4 position of the monosaccharide. The crystal structure of Gnk2 in complex with mannose revealed that three residues (asparagine-11, arginine-93, and glutamate-104) recognized mannose by hydrogen bonds, which defined the carbohydrate-binding specificity. These interactions were directly related to the ability of Gnk2 to inhibit the growth of fungi, including the plant pathogenic Fusarium spp., which were disrupted by mutation of arginine-93 or the presence of yeast mannan in the assay system. In addition, Gnk2 did not inhibit the growth of a yeast mutant strain lacking the α1,2-linked mannose moiety. These results provide insights into the molecular basis of the DUF26 protein family. PMID:25139159

  12. Ice Recrystallization in a Solution of a Cryoprotector and Its Inhibition by a Protein: Synchrotron X-Ray Diffraction Study.

    PubMed

    Zakharov, Boris; Fisyuk, Alexander; Fitch, Andy; Watier, Yves; Kostyuchenko, Anastasia; Varshney, Dushyant; Sztucki, Michael; Boldyreva, Elena; Shalaev, Evgenyi

    2016-07-01

    Ice formation and recrystallization is a key phenomenon in freezing and freeze-drying of pharmaceuticals and biopharmaceuticals. In this investigation, high-resolution synchrotron X-ray diffraction is used to quantify the extent of disorder of ice crystals in binary aqueous solutions of a cryoprotectant (sorbitol) and a protein, bovine serum albumin. Ice crystals in more dilute (10 wt%) solutions have lower level of microstrain and larger crystal domain size than these in more concentrated (40 wt%) solutions. Warming the sorbitol-water mixtures from 100 to 228 K resulted in partial ice melting, with simultaneous reduction in the microstrain and increase in crystallite size, that is, recrystallization. In contrast to sorbitol solutions, ice crystals in the BSA solutions preserved both the microstrain and smaller crystallite size on partial melting, demonstrating that BSA inhibits ice recrystallization. The results are consistent with BSA partitioning into quasi-liquid layer on ice crystals but not with a direct protein-ice interaction and protein sorption on ice surface. The study shows for the first time that a common (i.e., not-antifreeze) protein can have a major impact on ice recrystallization and also presents synchrotron X-ray diffraction as a unique tool for quantification of crystallinity and disorder in frozen aqueous systems.

  13. Perplexing cooperative folding and stability of a low-sequence complexity, polyproline 2 protein lacking a hydrophobic core.

    PubMed

    Gates, Zachary P; Baxa, Michael C; Yu, Wookyung; Riback, Joshua A; Li, Hui; Roux, Benoît; Kent, Stephen B H; Sosnick, Tobin R

    2017-02-13

    The burial of hydrophobic side chains in a protein core generally is thought to be the major ingredient for stable, cooperative folding. Here, we show that, for the snow flea antifreeze protein (sfAFP), stability and cooperativity can occur without a hydrophobic core, and without α-helices or β-sheets. sfAFP has low sequence complexity with 46% glycine and an interior filled only with backbone H-bonds between six polyproline 2 (PP2) helices. However, the protein folds in a kinetically two-state manner and is moderately stable at room temperature. We believe that a major part of the stability arises from the unusual match between residue-level PP2 dihedral angle bias in the unfolded state and PP2 helical structure in the native state. Additional stabilizing factors that compensate for the dearth of hydrophobic burial include shorter and stronger H-bonds, and increased entropy in the folded state. These results extend our understanding of the origins of cooperativity and stability in protein folding, including the balance between solvent and polypeptide chain entropies.

  14. Neofunctionalization of zona pellucida proteins enhances freeze-prevention in the eggs of Antarctic notothenioids

    NASA Astrophysics Data System (ADS)

    Cao, Lixue; Huang, Qiao; Wu, Zhichao; Cao, Dong-Dong; Ma, Zhanling; Xu, Qianghua; Hu, Peng; Fu, Yanxia; Shen, Yu; Chan, Jiulin; Zhou, Cong-Zhao; Zhai, Wanying; Chen, Liangbiao

    2016-10-01

    The mechanisms by which the eggs of the Antarctic notothenioid fishes avoid freezing are not fully understood. Zona pellucida proteins (ZPs) are constituents of the chorion which forms a protective matrix surrounding the egg. Here we report occurrence of freezing temperature-related gene expansion and acquisition of unusual ice melting-promoting (IMP) activity in a family of Antarctic notothenioid ZPs (AnnotoZPs). Members of AnnotoZPs are shown to bind with ice and non-colligatively depress the melting point of a solution in a range of 0.26 to 0.65 °C at a moderate concentration. Eggs of zebrafishes expressing an AnnotoZP transgene show improved melting point depression and enhanced survival in freezing conditions. Mutational analyses in a representative AnnotoZP indicate the ZP domain and patches of acidic residues are essential structures for the IMP activity. AnnotoZPs, therefore, represent a group of macromolecules that prevent freezing by a unique ZP-ice interaction mechanism distinct from the known antifreeze proteins.

  15. Neofunctionalization of zona pellucida proteins enhances freeze-prevention in the eggs of Antarctic notothenioids

    PubMed Central

    Cao, Lixue; Huang, Qiao; Wu, Zhichao; Cao, Dong-dong; Ma, Zhanling; Xu, Qianghua; Hu, Peng; Fu, Yanxia; Shen, Yu; Chan, Jiulin; Zhou, Cong-zhao; Zhai, Wanying; Chen, Liangbiao

    2016-01-01

    The mechanisms by which the eggs of the Antarctic notothenioid fishes avoid freezing are not fully understood. Zona pellucida proteins (ZPs) are constituents of the chorion which forms a protective matrix surrounding the egg. Here we report occurrence of freezing temperature-related gene expansion and acquisition of unusual ice melting-promoting (IMP) activity in a family of Antarctic notothenioid ZPs (AnnotoZPs). Members of AnnotoZPs are shown to bind with ice and non-colligatively depress the melting point of a solution in a range of 0.26 to 0.65 °C at a moderate concentration. Eggs of zebrafishes expressing an AnnotoZP transgene show improved melting point depression and enhanced survival in freezing conditions. Mutational analyses in a representative AnnotoZP indicate the ZP domain and patches of acidic residues are essential structures for the IMP activity. AnnotoZPs, therefore, represent a group of macromolecules that prevent freezing by a unique ZP–ice interaction mechanism distinct from the known antifreeze proteins. PMID:27698404

  16. Neofunctionalization of zona pellucida proteins enhances freeze-prevention in the eggs of Antarctic notothenioids.

    PubMed

    Cao, Lixue; Huang, Qiao; Wu, Zhichao; Cao, Dong-Dong; Ma, Zhanling; Xu, Qianghua; Hu, Peng; Fu, Yanxia; Shen, Yu; Chan, Jiulin; Zhou, Cong-Zhao; Zhai, Wanying; Chen, Liangbiao

    2016-10-04

    The mechanisms by which the eggs of the Antarctic notothenioid fishes avoid freezing are not fully understood. Zona pellucida proteins (ZPs) are constituents of the chorion which forms a protective matrix surrounding the egg. Here we report occurrence of freezing temperature-related gene expansion and acquisition of unusual ice melting-promoting (IMP) activity in a family of Antarctic notothenioid ZPs (AnnotoZPs). Members of AnnotoZPs are shown to bind with ice and non-colligatively depress the melting point of a solution in a range of 0.26 to 0.65 °C at a moderate concentration. Eggs of zebrafishes expressing an AnnotoZP transgene show improved melting point depression and enhanced survival in freezing conditions. Mutational analyses in a representative AnnotoZP indicate the ZP domain and patches of acidic residues are essential structures for the IMP activity. AnnotoZPs, therefore, represent a group of macromolecules that prevent freezing by a unique ZP-ice interaction mechanism distinct from the known antifreeze proteins.

  17. Purification, crystallization and preliminary X-ray analysis of the ligand-binding domain of human lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1)

    SciTech Connect

    Ishigaki, Tomoko; Ohki, Izuru; Oyama, Takuji; Machida, Sachiko; Morikawa, Kousuke; Tate, Shin-ichi

    2005-05-01

    Two different fragments of the ligand-binding domain of LOX-1, the major receptor for oxidized low-density lipoprotein (LDL) on endothelial cells, have been crystallized in different forms. Two different fragments of the ligand-binding domain of LOX-1, the major receptor for oxidized low-density lipoprotein (LDL) on endothelial cells, have been crystallized in different forms. One crystal form contains the disulfide-linked dimer, which is the form of the molecule present on the cell surface; the other contains a monomeric form of the receptor that lacks the cysteine residue necessary to form disulfide-linked homodimers. The crystal of the monomeric ligand-binding domain belongs to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 56.79, b = 67.57, c = 79.02 Å. The crystal of the dimeric form belongs to space group C2, with unit-cell parameters a = 70.86, b = 49.56, c = 76.73 Å, β = 98.59°. Data for the dimeric form of the LOX-1 ligand-binding domain have been collected to 2.4 Å. For the monomeric form of the ligand-binding domain, native, heavy-atom derivative and SeMet-derivative crystals have been obtained; their diffraction data have been measured to 3.0, 2.4 and 1.8 Å resolution, respectively.

  18. Losartan attenuates human monocyte-derived dendritic cell immune maturation via downregulation of lectin-like oxidized low-density lipoprotein receptor-1.

    PubMed

    Huang, Dong; Lu, Hao; Liu, Hongying; Yao, Kang; Sun, Aijun; Zou, Yunzeng; Ge, Junbo

    2012-08-01

    The angiotensin II receptor-1 blockers have generally been shown to have antiatherogenic effects, and dendritic cells (DCs) are the most efficient antigen presenting cells that play an active role in the development of atherosclerosis through inflammatory-immune responses. Here, we tested the hypothesis that the antiatherogenic effect of losartan, the first angiotensin II receptor-1 blockers, might partly be mediated by attenuating DCs maturation. In this study, we showed that oxidized low-density lipoprotein (oxLDL) and angiotensin II (Ang II) could induce the maturation of human monocyte-derived DCs, stimulate CD83, HLA-DR expressions and IL-12, interferon-gamma secretions and increase the capacity of DCs to stimulate T-cell proliferation, which were suppressed by losartan. OxLDL could promote the autocrine secretion of Ang II by DCs and upregulate the expressions of 3 scavenger receptors SR-A, CD36, and LOX-1. Losartan reduced oxLDL-induced LOX-1 expression but not SR-A and CD36 expressions. Ang II could only upregulate the LOX-1 expression, which was reduced by losartan. OxLDL- and Ang II-induced upregulation of CD83 and secretion of IL-12 were all attenuated by LOX-1 neutralizing antibody. In conclusion, losartan could attenuate the oxLDL- and Ang II-induced immune maturation of human monocyte-derived DCs partly through downregulation of the LOX-1 expression.

  19. Rapid discrimination of MHC class I and killer cell lectin-like receptor allele variants by high-resolution melt analysis.

    PubMed

    Lundgren, Alyssa; Kim, Sharon; Stadnisky, Michael D; Brown, Michael G

    2012-08-01

    Ly49G and H-2 class I D(k) molecules are critical to natural killer cell-mediated viral control. To examine their contributions in greater depth, we established NK gene complex (NKC)/Ly49 congenic strains and a novel genetic model defined by MHC class I D(k) disparity in congenic and transgenic mouse strains. Generation and maintenance of Ly49 and H-2 class I select strains require efficient and reproducible genotyping assays for highly polygenic and polymorphic sequences. Thus, we coupled gene- and allele-specific PCR with high-resolution melt (HRM) analysis to discriminate Ly49g and H-2 class I D and K alleles in select strains and in the F(2) and backcross hybrid offspring of different genetic crosses. We show that HRM typing for these critical immune response genes is fast, accurate, and dependable. We further demonstrate that H-2 class I D HRM typing is competent to detect and quantify transgene copy numbers in different mice with distinct genetic backgrounds. Our findings substantiate the utility and practicality of HRM genotyping for highly related genes and alleles, even those belonging to clustered multigene families. Based on these findings, we envision that HRM is capable to interrogate and quantify gene- and allele-specific variations due to differential regulation of gene expression.

  20. Cryopreservative effects of the recombinant ice-binding protein from the arctic yeast Leucosporidium sp. on red blood cells.

    PubMed

    Lee, Sung Gu; Koh, Hye Yeon; Lee, Jun Hyuck; Kang, Sung-Ho; Kim, Hak Jun

    2012-06-01

    Antifreeze proteins (AFPs) have important functions in many freeze-tolerant organisms. The proteins non-colligatively lower the freezing point and functionally inhibit ice recrystallization in frozen solutions. In our previous studies, we found that the Arctic yeast Leucosporidium sp. produces an AFP (LeIBP), and that the protein could be successfully produced in Pichia expression system. The present study showed that recombinant LeIBP possesses the ability to reduce the damage induced to red blood cells (RBCs) by freeze thawing. In addition to 40 % glycerol, both 0.4 and 0.8 mg/ml LeIBPs significantly reduced freeze-thaw-induced hemolysis at either rapid- (45 °C) or slow-warming (22 °C) temperatures. Post-thaw cell counts of the cryopreserved RBCs were dramatically enhanced, in particular, in 0.8 mg/ml LeIBP. Interestingly, the cryopreserved cells in the presence of LeIBP showed preserved cell size distribution. These results indicate that the ability of LeIBP to inhibit ice recrystallization helps the RBCs avoid critically damaging electrolyte concentrations, which are known as solution effects. Considering all these data, LeIBP can be thought of as a key component in improving RBC cryopreservation efficiency.

  1. Honey Glycoproteins Containing Antimicrobial Peptides, Jelleins of the Major Royal Jelly Protein 1, Are Responsible for the Cell Wall Lytic and Bactericidal Activities of Honey

    PubMed Central

    Brudzynski, Katrina; Sjaarda, Calvin

    2015-01-01

    We have recently identified the bacterial cell wall as the cellular target for honey antibacterial compounds; however, the chemical nature of these compounds remained to be elucidated. Using Concavalin A- affinity chromatography, we found that isolated glycoprotein fractions (glps), but not flow-through fractions, exhibited strong growth inhibitory and bactericidal properties. The glps possessed two distinct functionalities: (a) specific binding and agglutination of bacterial cells, but not rat erythrocytes and (b) non-specific membrane permeabilization of both bacterial cells and erythrocytes. The isolated glps induced concentration- and time-dependent changes in the cell shape of both E. coli and B. subtilis as visualized by light and SEM microscopy. The appearance of filaments and spheroplasts correlated with growth inhibition and bactericidal effects, respectively. The time-kill kinetics showed a rapid, >5-log10 reduction of viable cells within 15 min incubation at 1xMBC, indicating that the glps-induced damage of the cell wall was lethal. Unexpectedly, MALDI-TOF and electrospray quadrupole time of flight mass spectrometry, (ESI-Q-TOF-MS/MS) analysis of glps showed sequence identity with the Major Royal Jelly Protein 1 (MRJP1) precursor that harbors three antimicrobial peptides: Jelleins 1, 2, and 4. The presence of high-mannose structures explained the lectin-like activity of MRJP1, while the presence of Jelleins in MRJP1 may explain cell wall disruptions. Thus, the observed damages induced by the MRJP1 to the bacterial cell wall constitute the mechanism by which the antibacterial effects were produced. Antibacterial activity of MRJP1 glps directly correlated with the overall antibacterial activity of honey, suggesting that it is honey’s active principle responsible for this activity. PMID:25830314

  2. Honey glycoproteins containing antimicrobial peptides, Jelleins of the Major Royal Jelly Protein 1, are responsible for the cell wall lytic and bactericidal activities of honey.

    PubMed

    Brudzynski, Katrina; Sjaarda, Calvin

    2015-01-01

    We have recently identified the bacterial cell wall as the cellular target for honey antibacterial compounds; however, the chemical nature of these compounds remained to be elucidated. Using Concavalin A-affinity chromatography, we found that isolated glycoprotein fractions (glps), but not flow-through fractions, exhibited strong growth inhibitory and bactericidal properties. The glps possessed two distinct functionalities: (a) specific binding and agglutination of bacterial cells, but not rat erythrocytes and (b) non-specific membrane permeabilization of both bacterial cells and erythrocytes. The isolated glps induced concentration- and time-dependent changes in the cell shape of both E. coli and B. subtilis as visualized by light and SEM microscopy. The appearance of filaments and spheroplasts correlated with growth inhibition and bactericidal effects, respectively. The time-kill kinetics showed a rapid, >5-log10 reduction of viable cells within 15 min incubation at 1xMBC, indicating that the glps-induced damage of the cell wall was lethal. Unexpectedly, MALDI-TOF and electrospray quadrupole time of flight mass spectrometry, (ESI-Q-TOF-MS/MS) analysis of glps showed sequence identity with the Major Royal Jelly Protein 1 (MRJP1) precursor that harbors three antimicrobial peptides: Jelleins 1, 2, and 4. The presence of high-mannose structures explained the lectin-like activity of MRJP1, while the presence of Jelleins in MRJP1 may explain cell wall disruptions. Thus, the observed damages induced by the MRJP1 to the bacterial cell wall constitute the mechanism by which the antibacterial effects were produced. Antibacterial activity of MRJP1 glps directly correlated with the overall antibacterial activity of honey, suggesting that it is honey's active principle responsible for this activity.

  3. Pepsin-pancreatin protein hydrolysates from extruded amaranth inhibit markers of atherosclerosis in LPS-induced THP-1 macrophages-like human cells by reducing expression of proteins in LOX-1 signaling pathway

    PubMed Central

    2014-01-01

    Background Atherosclerosis is considered a progressive disease that affects arteries that bring blood to the heart, to the brain and to the lower end. It derives from endothelial dysfunction and inflammation, which play an important role in the thrombotic complications of atherosclerosis. Cardiovascular disease is the leading cause of death around the world and one factor that can contribute to its progression and prevention is diet. Our previous study found that amaranth hydrolysates inhibited LPS-induced inflammation in human and mouse macrophages by preventing activation of NF-κB signaling. Furthermore, extrusion improved the anti-inflammatory effect of amaranth protein hydrolysates in both cell lines, probably attributed to the production of bioactive peptides during processing. Therefore, the objective of this study was to compare the anti-atherosclerotic potential of pepsin-pancreatin hydrolysates from unprocessed and extruded amaranth in THP-1 lipopolysaccharide-induced human macrophages and suggest the mechanism of action. Results Unprocessed amaranth hydrolysate (UAH) and extruded amaranth hydrolysate (EAH) showed a significant reduction in the expression of interleukin-4 (IL-4) (69% and 100%, respectively), interleukin-6 (IL-6) (64% and 52%, respectively), interleukin-22 (IL-22) (55% and 70%, respectively). Likewise, UAH and EAH showed a reduction in the expression of monocyte-chemo attractant protein-1 (MCP-1) (35% and 42%, respectively), transferrin receptor-1 (TfR-1) (48% and 61%, respectively), granulocyte-macrophage colony-stimulating factor (GM-CSF) (59% and 63%, respectively), and tumor necrosis factor-α (TNF-α) (60% and 63%, respectively). Also, EAH reduced the expression of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) (27%), intracellular adhesion molecule-1 (ICAM-1) (28%) and matrix metalloproteinase-9 (MMP-9) (19%), important molecular markers in the atherosclerosis pathway. EAH, led to a reduction of 58, 52 and 79% for

  4. The Distribution of Lectins across the Phylum Nematoda: A Genome-Wide Search

    PubMed Central

    Bauters, Lander; Naalden, Diana; Gheysen, Godelieve

    2017-01-01

    Nematodes are a very diverse phylum that has adapted to nearly every ecosystem. They have developed specialized lifestyles, dividing the phylum into free-living, animal, and plant parasitic species. Their sheer abundance in numbers and presence in nearly every ecosystem make them the most prevalent animals on earth. In this research nematode-specific profiles were designed to retrieve predicted lectin-like domains from the sequence data of nematode genomes and transcriptomes. Lectins are carbohydrate-binding proteins that play numerous roles inside and outside the cell depending on their sugar specificity and associated protein domains. The sugar-binding properties of the retrieved lectin-like proteins were predicted in silico. Although most research has focused on C-type lectin-like, galectin-like, and calreticulin-like proteins in nematodes, we show that the lectin-like repertoire in nematodes is far more diverse. We focused on C-type lectins, which are abundantly present in all investigated nematode species, but seem to be far more abundant in free-living species. Although C-type lectin-like proteins are omnipresent in nematodes, we have shown that only a small part possesses the residues that are thought to be essential for carbohydrate binding. Curiously, hevein, a typical plant lectin domain not reported in animals before, was found in some nematode species. PMID:28054982

  5. The Distribution of Lectins across the Phylum Nematoda: A Genome-Wide Search.

    PubMed

    Bauters, Lander; Naalden, Diana; Gheysen, Godelieve

    2017-01-04

    Nematodes are a very diverse phylum that has adapted to nearly every ecosystem. They have developed specialized lifestyles, dividing the phylum into free-living, animal, and plant parasitic species. Their sheer abundance in numbers and presence in nearly every ecosystem make them the most prevalent animals on earth. In this research nematode-specific profiles were designed to retrieve predicted lectin-like domains from the sequence data of nematode genomes and transcriptomes. Lectins are carbohydrate-binding proteins that play numerous roles inside and outside the cell depending on their sugar specificity and associated protein domains. The sugar-binding properties of the retrieved lectin-like proteins were predicted in silico. Although most research has focused on C-type lectin-like, galectin-like, and calreticulin-like proteins in nematodes, we show that the lectin-like repertoire in nematodes is far more diverse. We focused on C-type lectins, which are abundantly present in all investigated nematode species, but seem to be far more abundant in free-living species. Although C-type lectin-like proteins are omnipresent in nematodes, we have shown that only a small part possesses the residues that are thought to be essential for carbohydrate binding. Curiously, hevein, a typical plant lectin domain not reported in animals before, was found in some nematode species.

  6. Protein Condensation

    NASA Astrophysics Data System (ADS)

    Gunton, James D.; Shiryayev, Andrey; Pagan, Daniel L.

    2007-09-01

    Preface; 1. Introduction; 2. Globular protein structure; 3. Experimental methods; 4. Thermodynamics and statistical mechanics; 5. Protein-protein interactions; 6. Theoretical studies of equilibrium; 7. Nucleation theory; 8. Experimental studies of nucleation; 9. Lysozyme; 10. Some other globular proteins; 11. Membrane proteins; 12. Crystallins and cataracts; 13. Sickle hemoglobin and sickle cell anemia; 14, Alzheimer's disease; Index.

  7. Protein Condensation

    NASA Astrophysics Data System (ADS)

    Gunton, James D.; Shiryayev, Andrey; Pagan, Daniel L.

    2014-07-01

    Preface; 1. Introduction; 2. Globular protein structure; 3. Experimental methods; 4. Thermodynamics and statistical mechanics; 5. Protein-protein interactions; 6. Theoretical studies of equilibrium; 7. Nucleation theory; 8. Experimental studies of nucleation; 9. Lysozyme; 10. Some other globular proteins; 11. Membrane proteins; 12. Crystallins and cataracts; 13. Sickle hemoglobin and sickle cell anemia; 14, Alzheimer's disease; Index.

  8. Distribution of cold adaptation proteins in microbial mats in Lake Joyce, Antarctica: Analysis of metagenomic data by using two bioinformatics tools.

    PubMed

    Koo, Hyunmin; Hakim, Joseph A; Fisher, Phillip R E; Grueneberg, Alexander; Andersen, Dale T; Bej, Asim K

    2016-01-01

    In this study, we report the distribution and abundance of cold-adaptation proteins in microbial mat communities in the perennially ice-covered Lake Joyce, located in the McMurdo Dry Valleys, Antarctica. We have used MG-RAST and R code bioinformatics tools on Illumina HiSeq2000 shotgun metagenomic data and compared the filtering efficacy of these two methods on cold-adaptation proteins. Overall, the abundance of cold-shock DEAD-box protein A (CSDA), antifreeze proteins (AFPs), fatty acid desaturase (FAD), trehalose synthase (TS), and cold-shock family of proteins (CSPs) were present in all mat samples at high, moderate, or low levels, whereas the ice nucleation protein (INP) was present only in the ice and bulbous mat samples at insignificant levels. Considering the near homogeneous temperature profile of Lake Joyce (0.08-0.29 °C), the distribution and abundance of these proteins across various mat samples predictively correlated with known functional attributes necessary for microbial communities to thrive in this ecosystem. The comparison of the MG-RAST and the R code methods showed dissimilar occurrences of the cold-adaptation protein sequences, though with insignificant ANOSIM (R = 0.357; p-value = 0.012), ADONIS (R(2) = 0.274; p-value = 0.03) and STAMP (p-values = 0.521-0.984) statistical analyses. Furthermore, filtering targeted sequences using the R code accounted for taxonomic groups by avoiding sequence redundancies, whereas the MG-RAST provided total counts resulting in a higher sequence output. The results from this study revealed for the first time the distribution of cold-adaptation proteins in six different types of microbial mats in Lake Joyce, while suggesting a simpler and more manageable user-defined method of R code, as compared to a web-based MG-RAST pipeline.

  9. NDR proteins

    PubMed Central

    Jones, Alan M

    2010-01-01

    N-myc downregulated (NDR) genes were discovered more than fifteen years ago. Indirect evidence support a role in tumor progression and cellular differentiation, but their biochemical function is still unknown. Our detailed analyses on Arabidopsis NDR proteins (deisgnated NDR-like, NDL) show their involvement in altering auxin transport, local auxin gradients and expression level of auxin transport proteins. Animal NDL proteins may be involved in membrane recycling of E-cadherin and effector for the small GTPase. In light of these findings, we hypothesize that NDL proteins regulate vesicular trafficking of auxin transport facilitator PIN proteins by biochemically alterating the local lipid environment of PIN proteins. PMID:20724844

  10. Proteins (image)

    MedlinePlus

    ... is an important nutrient that builds muscles and bones and provides energy. Protein can help with weight control because it helps you feel full and satisfied from your meals. The healthiest proteins are the leanest. This means ...

  11. Protein Structure

    ERIC Educational Resources Information Center

    Asmus, Elaine Garbarino

    2007-01-01

    Individual students model specific amino acids and then, through dehydration synthesis, a class of students models a protein. The students clearly learn amino acid structure, primary, secondary, tertiary, and quaternary structure in proteins and the nature of the bonds maintaining a protein's shape. This activity is fun, concrete, inexpensive and…

  12. Therapeutic proteins.

    PubMed

    Dimitrov, Dimiter S

    2012-01-01

    Protein-based therapeutics are highly successful in clinic and currently enjoy unprecedented recognition of their potential. More than 100 genuine and similar number of modified therapeutic proteins are approved for clinical use in the European Union and the USA with 2010 sales of US$108 bln; monoclonal antibodies (mAbs) accounted for almost half (48%) of the sales. Based on their pharmacological activity, they can be divided into five groups: (a) replacing a protein that is deficient or abnormal; (b) augmenting an existing pathway; (c) providing a novel function or activity; (d) interfering with a molecule or organism; and (e) delivering other compounds or proteins, such as a radionuclide, cytotoxic drug, or effector proteins. Therapeutic proteins can also be grouped based on their molecular types that include antibody-based drugs, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, engineered protein scaffolds, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytics. They can also be classified based on their molecular mechanism of activity as (a) binding non-covalently to target, e.g., mAbs; (b) affecting covalent bonds, e.g., enzymes; and (c) exerting activity without specific interactions, e.g., serum albumin. Most protein therapeutics currently on the market are recombinant and hundreds of them are in clinical trials for therapy of cancers, immune disorders, infections, and other diseases. New engineered proteins, including bispecific mAbs and multispecific fusion proteins, mAbs conjugated with small molecule drugs, and proteins with optimized pharmacokinetics, are currently under development. However, in the last several decades, there are no conceptually new methodological developments comparable, e.g., to genetic engineering leading to the development of recombinant therapeutic proteins. It appears that a paradigm change in methodologies and understanding of mechanisms is needed to overcome major

  13. Whey Protein

    MedlinePlus

    ... inflammation (polymyalgia rheumatica). Taking whey protein in a dairy product twice daily for 8 weeks does not improve muscle function, walking speed, or other movement tests in people with polymyalgia rheumatica. Other conditions. More evidence is needed to rate whey protein for these uses.

  14. A novel lectin domain-containing protein (LvCTLD) associated with response of the whiteleg shrimp Penaeus (Litopenaeus) vannamei to yellow head virus (YHV).

    PubMed

    Junkunlo, Kingkamon; Prachumwat, Anuphap; Tangprasittipap, Amornrat; Senapin, Saengchan; Borwornpinyo, Suparerk; Flegel, Timothy W; Sritunyalucksana, Kallaya

    2012-07-01

    When using mRNA from gills of normal whiteleg shrimp Penaeus (Litopenaeus) vannamei as the tester and mRNA from yellow head virus (YHV)-infected shrimp as the driver, subtractive suppression hybridization (SSH) revealed that a novel EST clone of 198 bp with a putative C-type lectin-like domain (CTLD) was downregulated in YHV-infected shrimp. The clone nucleotide sequence had 99% identity with one contig MGID1052359 (1,380 bp) reported in an EST database of P. vannamei, and the presence of this target in normal shrimp was confirmed by RT-PCR using primers designed from the MGID1052359 sequence. Analysis of the primary structure of the deduced amino acid (a.a.) sequence of the contig revealed a short portion (40 a.a. residues) at its N-terminus with high similarity to a low density lipoprotein receptor (LDLR) class A domain and another 152 a.a. residues at its C-terminus with high similarity to a C-type lectin domain. Thus, the clone was named LvCTLD and three recombinant proteins (LvCTLD, the LDLR domain and the CTLD domain) were synthesized in a bacterial system based on its sequence. An in vitro encapsulation assay revealed that Sepharose 4B beads coated with rLvCTLD were encapsulated by shrimp hemocytes and that melanization followed by 24 h post-encapsulation. The encapsulation activity of rLvCTLD was inhibited by 100 mM galactose, but not mannose or EDTA. In vivo injection of rLvCTLD or rLvCTLD plus YHV resulted in a significant elevation of PO activity in the hemolymph of the challenged shrimp when compared to shrimp injected with buffer, suggesting that rLvCTLD could activate the proPO system. An ELISA test revealed that rLvCTLD could bind to YHV particles in the presence of shrimp hemolymph. Phylogenetic analysis suggested that the LvCTLD sequence was more closely related to an antiviral gene found in Penaeus monodon (PmAV) than to other reported shrimp lectins. Taken together, we conclude that a novel shrimp LvCTLD is a host recognition molecule involved in

  15. The GroEL protein of Porphyromonas gingivalis regulates atherogenic phenomena in endothelial cells mediated by upregulating toll-like receptor 4 expression

    PubMed Central

    Huang, Chun-Yao; Shih, Chun-Ming; Tsao, Nai-Wen; Lin, Yi-Wen; Shih, Chun-Che; Chiang, Kuang-Hsing; Shyue, Song-Kun; Chang, Yu-Jia; Hsieh, Chi-Kun; Lin, Feng-Yen

    2016-01-01

    Porphyromonas gingivalis (P. gingivalis) is a bacterial species that causes periodontitis. GroEL from P. gingivalis may possess biological activity and may be involved in the destruction of periodontal tissues. However, it is unclear whether P. gingivalis GroEL enhances the appearance of atherogenic phenomena in endothelial cells and vessels. Here, we constructed recombinant GroEL from P. gingivalis to investigate its effects in human coronary artery endothelial cells (HCAECs) in vitro and on aortas of high-cholesterol (HC)-fed B57BL/6 and B57BL/6-Tlr4lps-del mice in vivo. The results showed that GroEL impaired tube-formation capacity under non-cytotoxic conditions in HCAECs. GroEL increased THP-1 cell/HCAEC adhesion by increasing the expression of intracellular adhesion molecule (ICAM)-1 and vascular adhesion molecule (VCAM)-1 in endothelial cells. Additionally, GroEL increased DiI-oxidized low density lipoprotein (oxLDL) uptake, which may be mediated by elevated lectin-like oxLDL receptor (LOX)-1 but not scavenger receptor expressed by endothelial cells (SREC) and scavenger receptor class B1 (SR-B1) expression. Furthermore, GroEL interacts with toll-like receptor 4 (TLR4) and plays a causal role in atherogenesis in HCAECs. Human antigen R (HuR), an RNA-binding protein with a high affinity for the 3’ untranslated region (3’UTR) of TLR4 mRNA, contributes to the up-regulation of TLR4 induced by GroEL in HCAECs. In a GroEL animal administration study, GroEL elevated ICAM-1, VCAM-1, LOX-1 and TLR4 expression in the aortas of HC diet-fed wild C57BL/6 but not C57BL/6-Tlr4lps-del mice. Taken together, our findings suggest that P. gingivalis GroEL may contribute to cardiovascular disorders by affecting TLR4 expression. PMID:27158334

  16. Total protein

    MedlinePlus

    ... 2016:chap 215. Read More Agammaglobulinemia Albumin - blood (serum) test Amino acids Antibody Burns Chronic Congenital nephrotic syndrome Fibrinogen blood test Glomerulonephritis Hemoglobin Liver disease Malabsorption Multiple myeloma Polycythemia vera Protein in diet ...

  17. Protein Crystallizability.

    PubMed

    Smialowski, Pawel; Wong, Philip

    2016-01-01

    Obtaining diffracting quality crystals remains a major challenge in protein structure research. We summarize and compare methods for selecting the best protein targets for crystallization, construct optimization and crystallization condition design. Target selection methods are divided into algorithms predicting the chance of successful progression through all stages of structural determination (from cloning to solving the structure) and those focusing only on the crystallization step. We tried to highlight pros and cons of different approaches examining the following aspects: data size, redundancy and representativeness, overfitting during model construction, and results evaluation. In summary, although in recent years progress was made and several sequence properties were reported to be relevant for crystallization, the successful prediction of protein crystallization behavior and selection of corresponding crystallization conditions continue to challenge structural researchers.

  18. Protein Crystallization

    NASA Technical Reports Server (NTRS)

    Chernov, Alexander A.

    2005-01-01

    Nucleation, growth and perfection of protein crystals will be overviewed along with crystal mechanical properties. The knowledge is based on experiments using optical and force crystals behave similar to inorganic crystals, though with a difference in orders of magnitude in growing parameters. For example, the low incorporation rate of large biomolecules requires up to 100 times larger supersaturation to grow protein, rather than inorganic crystals. Nucleation is often poorly reproducible, partly because of turbulence accompanying the mixing of precipitant with protein solution. Light scattering reveals fluctuations of molecular cluster size, its growth, surface energies and increased clustering as protein ages. Growth most often occurs layer-by-layer resulting in faceted crystals. New molecular layer on crystal face is terminated by a step where molecular incorporation occurs. Quantitative data on the incorporation rate will be discussed. Rounded crystals with molecularly disordered interfaces will be explained. Defects in crystals compromise the x-ray diffraction resolution crucially needed to find the 3D atomic structure of biomolecules. The defects are immobile so that birth defects stay forever. All lattice defects known for inorganics are revealed in protein crystals. Contribution of molecular conformations to lattice disorder is important, but not studied. This contribution may be enhanced by stress field from other defects. Homologous impurities (e.g., dimers, acetylated molecules) are trapped more willingly by a growing crystal than foreign protein impurities. The trapped impurities induce internal stress eliminated in crystals exceeding a critical size (part of mni for ferritin, lysozyme). Lesser impurities are trapped from stagnant, as compared to the flowing, solution. Freezing may induce much more defects unless quickly amorphysizing intracrystalline water.

  19. Recombinant protein production technology

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recombinant protein production is an important technology for antibody production, biochemical activity study, and structural determination during the post-genomic era. Limiting factors in recombinant protein production include low-level protein expression, protein precipitation, and loss of protein...

  20. Antifreezes act as catalysts for methane hydrate formation from ice.

    PubMed

    McLaurin, Graham; Shin, Kyuchul; Alavi, Saman; Ripmeester, John A

    2014-09-22

    Contrary to the thermodynamic inhibiting effect of methanol on methane hydrate formation from aqueous phases, hydrate forms quickly at high yield by exposing frozen water-methanol mixtures with methanol concentrations ranging from 0.6-10 wt% to methane gas at pressures from 125 bars at 253 K. Formation rates are some two orders of magnitude greater than those obtained for samples without methanol and conversion of ice is essentially complete. Ammonia has a similar catalytic effect when used in concentrations of 0.3-2.7 wt%. The structure I methane hydrate formed in this manner was characterized by powder X-ray diffraction and Raman spectroscopy. Steps in the possible mechanism of action of methanol were studied with molecular dynamics simulations of the Ih (0001) basal plane exposed to methanol and methane gas. Simulations show that methanol from a surface aqueous layer slowly migrates into the ice lattice. Methane gas is preferentially adsorbed into the aqueous methanol surface layer. Possible consequences of the catalytic methane hydrate formation on hydrate plug formation in gas pipelines, on large scale energy-efficient gas hydrate formation, and in planetary science are discussed.

  1. Protein inference: A protein quantification perspective.

    PubMed

    He, Zengyou; Huang, Ting; Liu, Xiaoqing; Zhu, Peijun; Teng, Ben; Deng, Shengchun

    2016-08-01

    In mass spectrometry-based shotgun proteomics, protein quantification and protein identification are two major computational problems. To quantify the protein abundance, a list of proteins must be firstly inferred from the raw data. Then the relative or absolute protein abundance is estimated with quantification methods, such as spectral counting. Until now, most researchers have been dealing with these two processes separately. In fact, the protein inference problem can be regarded as a special protein quantification problem in the sense that truly present proteins are those proteins whose abundance values are not zero. Some recent published papers have conceptually discussed this possibility. However, there is still a lack of rigorous experimental studies to test this hypothesis. In this paper, we investigate the feasibility of using protein quantification methods to solve the protein inference problem. Protein inference methods aim to determine whether each candidate protein is present in the sample or not. Protein quantification methods estimate the abundance value of each inferred protein. Naturally, the abundance value of an absent protein should be zero. Thus, we argue that the protein inference problem can be viewed as a special protein quantification problem in which one protein is considered to be present if its abundance is not zero. Based on this idea, our paper tries to use three simple protein quantification methods to solve the protein inference problem effectively. The experimental results on six data sets show that these three methods are competitive with previous protein inference algorithms. This demonstrates that it is plausible to model the protein inference problem as a special protein quantification task, which opens the door of devising more effective protein inference algorithms from a quantification perspective. The source codes of our methods are available at: http://code.google.com/p/protein-inference/.

  2. Learning about Proteins

    MedlinePlus

    ... What Happens in the Operating Room? Learning About Proteins KidsHealth > For Kids > Learning About Proteins A A ... the foods you eat. continue Different Kinds of Protein Protein from animal sources, such as meat and ...

  3. Protein Microarray Technology

    PubMed Central

    Hall, David A.; Ptacek, Jason

    2007-01-01

    Protein chips have emerged as a promising approach for a wide variety of applications including the identification of protein-protein interactions, protein-phospholipid interactions, small molecule targets, and substrates of proteins kinases. They can also be used for clinical diagnostics and monitoring disease states. This article reviews current methods in the generation and applications of protein microarrays. PMID:17126887

  4. Length, protein protein interactions, and complexity

    NASA Astrophysics Data System (ADS)

    Tan, Taison; Frenkel, Daan; Gupta, Vishal; Deem, Michael W.

    2005-05-01

    The evolutionary reason for the increase in gene length from archaea to prokaryotes to eukaryotes observed in large-scale genome sequencing efforts has been unclear. We propose here that the increasing complexity of protein-protein interactions has driven the selection of longer proteins, as they are more able to distinguish among a larger number of distinct interactions due to their greater average surface area. Annotated protein sequences available from the SWISS-PROT database were analyzed for 13 eukaryotes, eight bacteria, and two archaea species. The number of subcellular locations to which each protein is associated is used as a measure of the number of interactions to which a protein participates. Two databases of yeast protein-protein interactions were used as another measure of the number of interactions to which each S. cerevisiae protein participates. Protein length is shown to correlate with both number of subcellular locations to which a protein is associated and number of interactions as measured by yeast two-hybrid experiments. Protein length is also shown to correlate with the probability that the protein is encoded by an essential gene. Interestingly, average protein length and number of subcellular locations are not significantly different between all human proteins and protein targets of known, marketed drugs. Increased protein length appears to be a significant mechanism by which the increasing complexity of protein-protein interaction networks is accommodated within the natural evolution of species. Consideration of protein length may be a valuable tool in drug design, one that predicts different strategies for inhibiting interactions in aberrant and normal pathways.

  5. EDITORIAL: Precision proteins Precision proteins

    NASA Astrophysics Data System (ADS)

    Demming, Anna

    2010-06-01

    Since the birth of modern day medicine, during the times of Hippocrates in ancient Greece, the profession has developed from the rudimentary classification of disease into a rigorous science with an inspiring capability to treat and cure. Scientific methodology has distilled clinical diagnostic tools from the early arts of prognosis, which used to rely as much on revelation and prophecy, as intuition and judgement [1]. Over the past decade, research into the interactions between proteins and nanosystems has provided some ingenious and apt techniques for delving into the intricacies of anatomical systems. In vivo biosensing has emerged as a vibrant field of research, as much of medical diagnosis relies on the detection of substances or an imbalance in the chemicals in the body. The inherent properties of nanoscale structures, such as cantilevers, make them well suited to biosensing applications that demand the detection of molecules at very low concentrations. Measurable deflections in cantilevers functionalised with antibodies provide quantitative indicators of the presence of specific antigens when the two react. Such developments have roused mounting interest in the interactions of proteins with nanostructures, such as carbon nanotubes [3], which have demonstrated great potential as generic biomarkers. Plasmonic properties are also being exploited in sensing applications, such as the molecular sentinel recently devised by researchers in the US. The device uses the plasmonic properties of a silver nanoparticle linked to a Raman labelled hairpin DNA probe to signal changes in the probe geometry resulting from interactions with substances in the environment. Success stories so far include the detection of two specific genes associated with breast cancer [4]. A greater understanding of how RNA interference regulates gene expression has highlighted the potential of using this natural process as another agent for combating disease in personalized medicine. However, the

  6. Shotgun protein sequencing.

    SciTech Connect

    Faulon, Jean-Loup Michel; Heffelfinger, Grant S.

    2009-06-01

    A novel experimental and computational technique based on multiple enzymatic digestion of a protein or protein mixture that reconstructs protein sequences from sequences of overlapping peptides is described in this SAND report. This approach, analogous to shotgun sequencing of DNA, is to be used to sequence alternative spliced proteins, to identify post-translational modifications, and to sequence genetically engineered proteins.

  7. Protein Crystal Based Nanomaterials

    NASA Technical Reports Server (NTRS)

    Bell, Jeffrey A.; VanRoey, Patrick

    2001-01-01

    This is the final report on a NASA Grant. It concerns a description of work done, which includes: (1) Protein crystals cross-linked to form fibers; (2) Engineering of protein to favor crystallization; (3) Better knowledge-based potentials for protein-protein contacts; (4) Simulation of protein crystallization.

  8. Protein-losing enteropathy

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/007338.htm Protein-losing enteropathy To use the sharing features on this page, please enable JavaScript. Protein-losing enteropathy is an abnormal loss of protein ...

  9. Protein in diet

    MedlinePlus

    ... basic structure of protein is a chain of amino acids. You need protein in your diet to help ... Protein foods are broken down into parts called amino acids during digestion. The human body needs a number ...

  10. Protein splicing: selfish genes invade cellular proteins.

    PubMed

    Neff, N F

    1993-12-01

    Protein splicing is a series of enzymatic events involving intramolecular protein breakage, rejoining and intron homing, in which introns are able to promote the recombinative transposition of their own coding sequences. Eukaryotic and prokaryotic spliced proteins have conserved similar gene structure, but little amino acid identity. The genes coding for these spliced proteins contain internal in-frame introns that encode polypeptides that apparently self-excise from the resulting host protein sequences. Excision of the 'protein intron' is coupled with joining of the two flanking protein regions encoded by exons of the host gene. Some introns of this type encode DNA endonucleases, related to Group I RNA intron gene products, that stimulate gene conversion and self-transmission.

  11. Targeted expression of redesigned and codon optimised synthetic gene leads to recrystallisation inhibition and reduced electrolyte leakage in spring wheat at sub-zero temperatures.

    PubMed

    Khanna, Harjeet K; Daggard, Grant E

    2006-12-01

    Antifreeze proteins (AFPs) adsorb to ice crystals and inhibit their growth, leading to non-colligative freezing point depression. Crops like spring wheat, that are highly susceptible to frost damage, can potentially be made frost tolerant by expressing AFPs in the cytoplasm and apoplast where ice recrystallisation leads to cellular damage. The protein sequence for HPLC-6 alpha-helical antifreeze protein from winter flounder was rationally redesigned after removing the prosequences in the native protein. Wheat nuclear gene preferred amino acid codons were used to synthesize a recombinant antifreeze gene, rAFPI. Antifreeze protein was targeted to the apoplast using a Murine leader peptide sequence from the mAb24 light chain or retained in the endoplasmic reticulum using C-terminus KDEL sequence. The coding sequences were placed downstream of the rice Actin promoter and Actin-1 intron and upstream of the nopaline synthase terminator in the plant expression vectors. Transgenic wheat lines were generated through micro projectile bombardment of immature embryos of spring wheat cultivar Seri 82. Levels of antifreeze protein in the transgenic lines without any targeting peptide were low (0.06-0.07%). The apoplast-targeted protein reached a level of 1.61% of total soluble protein, 90% of which was present in the apoplast. ER-retained protein accumulated in the cells at levels up to 0.65% of total soluble proteins. Transgenic wheat line T-8 with apoplast-targeted antifreeze protein exhibited the highest levels of antifreeze activity and provided significant freezing protection even at temperatures as low as -7 degrees C.

  12. PREFACE: Protein protein interactions: principles and predictions

    NASA Astrophysics Data System (ADS)

    Nussinov, Ruth; Tsai, Chung-Jung

    2005-06-01

    Proteins are the `workhorses' of the cell. Their roles span functions as diverse as being molecular machines and signalling. They carry out catalytic reactions, transport, form viral capsids, traverse membranes and form regulated channels, transmit information from DNA to RNA, making possible the synthesis of new proteins, and they are responsible for the degradation of unnecessary proteins and nucleic acids. They are the vehicles of the immune response and are responsible for viral entry into the cell. Given their importance, considerable effort has been centered on the prediction of protein function. A prime way to do this is through identification of binding partners. If the function of at least one of the components with which the protein interacts is known, that should let us assign its function(s) and the pathway(s) in which it plays a role. This holds since the vast majority of their chores in the living cell involve protein-protein interactions. Hence, through the intricate network of these interactions we can map cellular pathways, their interconnectivities and their dynamic regulation. Their identification is at the heart of functional genomics; their prediction is crucial for drug discovery. Knowledge of the pathway, its topology, length, and dynamics may provide useful information for forecasting side effects. The goal of predicting protein-protein interactions is daunting. Some associations are obligatory, others are continuously forming and dissociating. In principle, from the physical standpoint, any two proteins can interact, but under what conditions and at which strength? The principles of protein-protein interactions are general: the non-covalent interactions of two proteins are largely the outcome of the hydrophobic effect, which drives the interactions. In addition, hydrogen bonds and electrostatic interactions play important roles. Thus, many of the interactions observed in vitro are the outcome of experimental overexpression. Protein disorder

  13. Protein sequence comparison and protein evolution

    SciTech Connect

    Pearson, W.R.

    1995-12-31

    This tutorial was one of eight tutorials selected to be presented at the Third International Conference on Intelligent Systems for Molecular Biology which was held in the United Kingdom from July 16 to 19, 1995. This tutorial examines how the information conserved during the evolution of a protein molecule can be used to infer reliably homology, and thus a shared proteinfold and possibly a shared active site or function. The authors start by reviewing a geological/evolutionary time scale. Next they look at the evolution of several protein families. During the tutorial, these families will be used to demonstrate that homologous protein ancestry can be inferred with confidence. They also examine different modes of protein evolution and consider some hypotheses that have been presented to explain the very earliest events in protein evolution. The next part of the tutorial will examine the technical aspects of protein sequence comparison. Both optimal and heuristic algorithms and their associated parameters that are used to characterize protein sequence similarities are discussed. Perhaps more importantly, they survey the statistics of local similarity scores, and how these statistics can both be used to improve the selectivity of a search and to evaluate the significance of a match. They them examine distantly related members of three protein families, the serine proteases, the glutathione transferases, and the G-protein-coupled receptors (GCRs). Finally, the discuss how sequence similarity can be used to examine internal repeated or mosaic structures in proteins.

  14. Whey protein fractionation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Concentrated whey protein products from cheese whey, such as whey protein concentrate (WPC) and whey protein isolate (WPI), contain more than seven different types of proteins: alpha-lactalbumin (alpha-LA), beta-lactoglobulin (beta-LG), bovine serum albumin (BSA), immunoglobulins (Igs), lactoferrin ...

  15. Protein- protein interaction detection system using fluorescent protein microdomains

    DOEpatents

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2010-02-23

    The invention provides a protein labeling and interaction detection system based on engineered fragments of fluorescent and chromophoric proteins that require fused interacting polypeptides to drive the association of the fragments, and further are soluble and stable, and do not change the solubility of polypeptides to which they are fused. In one embodiment, a test protein X is fused to a sixteen amino acid fragment of GFP (.beta.-strand 10, amino acids 198-214), engineered to not perturb fusion protein solubility. A second test protein Y is fused to a sixteen amino acid fragment of GFP (.beta.-strand 11, amino acids 215-230), engineered to not perturb fusion protein solubility. When X and Y interact, they bring the GFP strands into proximity, and are detected by complementation with a third GFP fragment consisting of GFP amino acids 1-198 (strands 1-9). When GFP strands 10 and 11 are held together by interaction of protein X and Y, they spontaneous association with GFP strands 1-9, resulting in structural complementation, folding, and concomitant GFP fluorescence.

  16. Molecular modelling of protein-protein/protein-solvent interactions

    NASA Astrophysics Data System (ADS)

    Luchko, Tyler

    The inner workings of individual cells are based on intricate networks of protein-protein interactions. However, each of these individual protein interactions requires a complex physical interaction between proteins and their aqueous environment at the atomic scale. In this thesis, molecular dynamics simulations are used in three theoretical studies to gain insight at the atomic scale about protein hydration, protein structure and tubulin-tubulin (protein-protein) interactions, as found in microtubules. Also presented, in a fourth project, is a molecular model of solvation coupled with the Amber molecular modelling package, to facilitate further studies without the need of explicitly modelled water. Basic properties of a minimally solvated protein were calculated through an extended study of myoglobin hydration with explicit solvent, directly investigating water and protein polarization. Results indicate a close correlation between polarization of both water and protein and the onset of protein function. The methodology of explicit solvent molecular dynamics was further used to study tubulin and microtubules. Extensive conformational sampling of the carboxy-terminal tails of 8-tubulin was performed via replica exchange molecular dynamics, allowing the characterisation of the flexibility, secondary structure and binding domains of the C-terminal tails through statistical analysis methods. Mechanical properties of tubulin and microtubules were calculated with adaptive biasing force molecular dynamics. The function of the M-loop in microtubule stability was demonstrated in these simulations. The flexibility of this loop allowed constant contacts between the protofilaments to be maintained during simulations while the smooth deformation provided a spring-like restoring force. Additionally, calculating the free energy profile between the straight and bent tubulin configurations was used to test the proposed conformational change in tubulin, thought to cause microtubule

  17. Surface Mediated Protein Disaggregation

    NASA Astrophysics Data System (ADS)

    Radhakrishna, Mithun; Kumar, Sanat K.

    2014-03-01

    Preventing protein aggregation is of both biological and industrial importance. Biologically these aggregates are known to cause amyloid type diseases like Alzheimer's and Parkinson's disease. Protein aggregation leads to reduced activity of the enzymes in industrial applications. Inter-protein interactions between the hydrophobic residues of the protein are known to be the major driving force for protein aggregation. In the current paper we show how surface chemistry and curvature can be tuned to mitigate these inter-protein interactions. Our results calculated in the framework of the Hydrophobic-Polar (HP) lattice model show that, inter-protein interactions can be drastically reduced by increasing the surface hydrophobicity to a critical value corresponding to the adsorption transition of the protein. At this value of surface hydrophobicity, proteins lose inter-protein contacts to gain surface contacts and thus the surface helps in reducing the inter-protein interactions. Further, we show that the adsorption of the proteins inside hydrophobic pores of optimal sizes are most efficient both in reducing inter-protein contacts and simultaneously retaining most of the native-contacts due to strong protein-surface interactions coupled with stabilization due to the confinement. Department of Energy (Grant No DE-FG02-11ER46811).

  18. Physics of protein motility and motor proteins

    NASA Astrophysics Data System (ADS)

    Kolomeisky, Anatoly B.

    2013-09-01

    Motor proteins are enzymatic molecules that transform chemical energy into mechanical motion and work. They are critically important for supporting various cellular activities and functions. In the last 15 years significant progress in understanding the functioning of motor proteins has been achieved due to revolutionary breakthroughs in single-molecule experimental techniques and strong advances in theoretical modelling. However, microscopic mechanisms of protein motility are still not well explained, and the collective efforts of many scientists are needed in order to solve these complex problems. In this special section the reader will find the latest advances on the difficult road to mapping motor proteins dynamics in various systems. Recent experimental developments have allowed researchers to monitor and to influence the activity of single motor proteins with a high spatial and temporal resolution. It has stimulated significant theoretical efforts to understand the non-equilibrium nature of protein motility phenomena. The latest results from all these advances are presented and discussed in this special section. We would like to thank the scientists from all over the world who have reported their latest research results for this special section. We are also grateful to the staff and editors of Journal of Physics: Condensed Matter for their invaluable help in handling all the administrative and refereeing activities. The field of motor proteins and protein motility is fast moving, and we hope that this collection of articles will be a useful source of information in this highly interdisciplinary area. Physics of protein motility and motor proteins contents Physics of protein motility and motor proteinsAnatoly B Kolomeisky Identification of unique interactions between the flexible linker and the RecA-like domains of DEAD-box helicase Mss116 Yuan Zhang, Mirkó Palla, Andrew Sun and Jung-Chi Liao The load dependence of the physical properties of a molecular motor

  19. Protein C blood test

    MedlinePlus

    ... a normal substance in the body that prevents blood clotting. A blood test can be done to see ... history of blood clots. Protein C helps control blood clotting. A lack of this protein or problem with ...

  20. Protein S blood test

    MedlinePlus

    ... a normal substance in your body that prevents blood clotting. A blood test can be done to see ... family history of blood clots. Protein S helps control blood clotting. A lack of this protein or problem with ...

  1. Learning about Proteins

    MedlinePlus

    ... body, and protecting you from disease. All About Amino Acids When you eat foods that contain protein, the ... called amino (say: uh-MEE-no) acids. The amino acids then can be reused to make the proteins ...

  2. Modeling Protein Self Assembly

    ERIC Educational Resources Information Center

    Baker, William P.; Jones, Carleton Buck; Hull, Elizabeth

    2004-01-01

    Understanding the structure and function of proteins is an important part of the standards-based science curriculum. Proteins serve vital roles within the cell and malfunctions in protein self assembly are implicated in degenerative diseases. Experience indicates that this topic is a difficult one for many students. We have found that the concept…

  3. CSF total protein

    MedlinePlus

    CSF total protein is a test to determine the amount of protein in your spinal fluid, also called cerebrospinal fluid (CSF). ... The normal protein range varies from lab to lab, but is typically about 15 to 60 milligrams per deciliter (mg/dL) ...

  4. Modeling Protein Domain Function

    ERIC Educational Resources Information Center

    Baker, William P.; Jones, Carleton "Buck"; Hull, Elizabeth

    2007-01-01

    This simple but effective laboratory exercise helps students understand the concept of protein domain function. They use foam beads, Styrofoam craft balls, and pipe cleaners to explore how domains within protein active sites interact to form a functional protein. The activity allows students to gain content mastery and an understanding of the…

  5. Destabilized bioluminescent proteins

    DOEpatents

    Allen, Michael S.; Rakesh, Gupta; Gary, Sayler S.

    2007-07-31

    Purified nucleic acids, vectors and cells containing a gene cassette encoding at least one modified bioluminescent protein, wherein the modification includes the addition of a peptide sequence. The duration of bioluminescence emitted by the modified bioluminescent protein is shorter than the duration of bioluminescence emitted by an unmodified form of the bioluminescent protein.

  6. Texturized dairy proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Dairy proteins are amenable to structural modifications induced by high temperature, shear and moisture; in particular, whey proteins can change conformation to new unfolded states. The change in protein state is a basis for creating new foods. The dairy products, nonfat dried milk (NDM), whey prote...

  7. Overview of Protein Microarrays

    PubMed Central

    Reymond Sutandy, FX; Qian, Jiang; Chen, Chien-Sheng; Zhu, Heng

    2013-01-01

    Protein microarray is an emerging technology that provides a versatile platform for characterization of hundreds of thousands of proteins in a highly parallel and high-throughput way. Two major classes of protein microarrays are defined to describe their applications: analytical and functional protein microarrays. In addition, tissue or cell lysates can also be fractionated and spotted on a slide to form a reverse-phase protein microarray. While the fabrication technology is maturing, applications of protein microarrays, especially functional protein microarrays, have flourished during the past decade. Here, we will first review recent advances in the protein microarray technologies, and then present a series of examples to illustrate the applications of analytical and functional protein microarrays in both basic and clinical research. The research areas will include detection of various binding properties of proteins, study of protein posttranslational modifications, analysis of host-microbe interactions, profiling antibody specificity, and identification of biomarkers in autoimmune diseases. As a powerful technology platform, it would not be surprising if protein microarrays will become one of the leading technologies in proteomic and diagnostic fields in the next decade. PMID:23546620

  8. The E5 Proteins

    PubMed Central

    DiMaio, Daniel; Petti, Lisa

    2013-01-01

    The E5 proteins are short transmembrane proteins encoded by many animal and human papillomaviruses. These proteins display transforming activity in cultured cells and animals, and they presumably also play a role in the productive virus life cycle. The E5 proteins are thought to act by modulating the activity of cellular proteins. Here, we describe the biological activities of the best-studied E5 proteins and discuss the evidence implicating specific protein targets and pathways in mediating these activities. The primary target of the 44-amino acid BPV1 E5 is the PDGF β receptor, whereas the EGF receptor appears to be an important target of the 83-amino acid HPV16 E5 protein. Both E5 proteins also bind to the vacuolar ATPase and affect MHC class I expression and cell-cell communication. Continued studies of the E5 proteins will elucidate important aspects of transmembrane protein-protein interactions, cellular signal transduction, cell biology, virus replication, and tumorigenesis. PMID:23731971

  9. Protopia: a protein-protein interaction tool

    PubMed Central

    Real-Chicharro, Alejandro; Ruiz-Mostazo, Iván; Navas-Delgado, Ismael; Kerzazi, Amine; Chniber, Othmane; Sánchez-Jiménez, Francisca; Medina, Miguel Ángel; Aldana-Montes, José F

    2009-01-01

    Background Protein-protein interactions can be considered the basic skeleton for living organism self-organization and homeostasis. Impressive quantities of experimental data are being obtained and computational tools are essential to integrate and to organize this information. This paper presents Protopia, a biological tool that offers a way of searching for proteins and their interactions in different Protein Interaction Web Databases, as a part of a multidisciplinary initiative of our institution for the integration of biological data . Results The tool accesses the different Databases (at present, the free version of Transfac, DIP, Hprd, Int-Act and iHop), and results are expressed with biological protein names or databases codes and can be depicted as a vector or a matrix. They can be represented and handled interactively as an organic graph. Comparison among databases is carried out using the Uniprot codes annotated for each protein. Conclusion The tool locates and integrates the current information stored in the aforementioned databases, and redundancies among them are detected. Results are compatible with the most important network analysers, so that they can be compared and analysed by other world-wide known tools and platforms. The visualization possibilities help to attain this goal and they are especially interesting for handling multiple-step or complex networks. PMID:19828077

  10. Protein-protein interactions in multienzyme megasynthetases.

    PubMed

    Weissman, Kira J; Müller, Rolf

    2008-04-14

    The multienzyme polyketide synthases (PKSs), nonribosomal polypeptide synthetases (NRPSs), and their hybrids are responsible for the construction in bacteria of numerous natural products of clinical value. These systems generate high structural complexity by using a simple biosynthetic logic--that of the assembly line. Each of the individual steps in building the metabolites is designated to an independently folded domain within gigantic polypeptides. The domains are clustered into functional modules, and the modules are strung out along the proteins in the order in which they act. Every metabolite results, therefore, from the successive action of up to 100 individual catalysts. Despite the conceptual simplicity of this division-of-labor organization, we are only beginning to decipher the molecular details of the numerous protein-protein interactions that support assembly-line biosynthesis, and which are critical to attempts to re-engineer these systems as a tool in drug discovery. This review aims to summarize the state of knowledge about several aspects of protein-protein interactions, including current architectural models for PKS and NRPS systems, the central role of carrier proteins, and the structural basis for intersubunit recognition.

  11. Highly thermostable fluorescent proteins

    DOEpatents

    Bradbury, Andrew M [Santa Fe, NM; Waldo, Geoffrey S [Santa Fe, NM; Kiss, Csaba [Los Alamos, NM

    2012-05-01

    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  12. Highly thermostable fluorescent proteins

    DOEpatents

    Bradbury, Andrew M.; Waldo, Geoffrey S.; Kiss, Csaba

    2011-03-22

    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  13. Highly thermostable fluorescent proteins

    DOEpatents

    Bradbury, Andrew M [Santa Fe, NM; Waldo, Geoffrey S [Santa Fe, NM; Kiss, Csaba [Los Alamos, NM

    2011-11-29

    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  14. Protein crystallization with paper

    NASA Astrophysics Data System (ADS)

    Matsuoka, Miki; Kakinouchi, Keisuke; Adachi, Hiroaki; Maruyama, Mihoko; Sugiyama, Shigeru; Sano, Satoshi; Yoshikawa, Hiroshi Y.; Takahashi, Yoshinori; Yoshimura, Masashi; Matsumura, Hiroyoshi; Murakami, Satoshi; Inoue, Tsuyoshi; Mori, Yusuke; Takano, Kazufumi

    2016-05-01

    We developed a new protein crystallization method that incorporates paper. A small piece of paper, such as facial tissue or KimWipes, was added to a drop of protein solution in the traditional sitting drop vapor diffusion technique, and protein crystals grew by incorporating paper. By this method, we achieved the growth of protein crystals with reducing osmotic shock. Because the technique is very simple and the materials are easy to obtain, this method will come into wide use for protein crystallization. In the future, it could be applied to nanoliter-scale crystallization screening on a paper sheet such as in inkjet printing.

  15. [Atypical ubiquitination of proteins].

    PubMed

    Buneeva, O A; Medvedev, A E

    2016-07-01

    Ubiquitination is a type of posttranslational modification of intracellular proteins characterized by covalent attachment of one (monoubiquitination) or several (polyubiquitination) of ubiquitin molecules to target proteins. In the case of polyubiquitination, linear or branched polyubiquitin chains are formed. Their formation involves various lysine residues of monomeric ubiquitin. The best studied is Lys48-polyubiquitination, which targets proteins for proteasomal degradation. In this review we have considered examples of so-called atypical polyubiquitination, which mainly involves other lysine residues (Lys6, Lys11, Lys27, Lys29, Lys33, Lys63) and also N-terminal methionine. The considered examples convincingly demonstrate that polyubiquitination of proteins not necessarily targets proteins for their proteolytic degradation in proteasomes. Atypically polyubiquitinated proteins are involved in regulation of various processes and altered polyubiquitination of certain proteins is crucial for development of serious diseases.

  16. Protein and vegetarian diets.

    PubMed

    Marsh, Kate A; Munn, Elizabeth A; Baines, Surinder K

    2013-08-19

    A vegetarian diet can easily meet human dietary protein requirements as long as energy needs are met and a variety of foods are eaten. Vegetarians should obtain protein from a variety of plant sources, including legumes, soy products, grains, nuts and seeds. Eggs and dairy products also provide protein for those following a lacto-ovo-vegetarian diet. There is no need to consciously combine different plant proteins at each meal as long as a variety of foods are eaten from day to day, because the human body maintains a pool of amino acids which can be used to complement dietary protein. The consumption of plant proteins rather than animal proteins by vegetarians may contribute to their reduced risk of chronic diseases such as diabetes and heart disease.

  17. Protein solubility modeling

    NASA Technical Reports Server (NTRS)

    Agena, S. M.; Pusey, M. L.; Bogle, I. D.

    1999-01-01

    A thermodynamic framework (UNIQUAC model with temperature dependent parameters) is applied to model the salt-induced protein crystallization equilibrium, i.e., protein solubility. The framework introduces a term for the solubility product describing protein transfer between the liquid and solid phase and a term for the solution behavior describing deviation from ideal solution. Protein solubility is modeled as a function of salt concentration and temperature for a four-component system consisting of a protein, pseudo solvent (water and buffer), cation, and anion (salt). Two different systems, lysozyme with sodium chloride and concanavalin A with ammonium sulfate, are investigated. Comparison of the modeled and experimental protein solubility data results in an average root mean square deviation of 5.8%, demonstrating that the model closely follows the experimental behavior. Model calculations and model parameters are reviewed to examine the model and protein crystallization process. Copyright 1999 John Wiley & Sons, Inc.

  18. Predictions of Protein-Protein Interfaces within Membrane Protein Complexes

    PubMed Central

    Asadabadi, Ebrahim Barzegari; Abdolmaleki, Parviz

    2013-01-01

    Background Prediction of interaction sites within the membrane protein complexes using the sequence data is of a great importance, because it would find applications in modification of molecules transport through membrane, signaling pathways and drug targets of many diseases. Nevertheless, it has gained little attention from the protein structural bioinformatics community. Methods In this study, a wide variety of prediction and classification tools were applied to distinguish the residues at the interfaces of membrane proteins from those not in the interfaces. Results The tuned SVM model achieved the high accuracy of 86.95% and the AUC of 0.812 which outperforms the results of the only previous similar study. Nevertheless, prediction performances obtained using most employed models cannot be used in applied fields and needs more effort to improve. Conclusion Considering the variety of the applied tools in this study, the present investigation could be a good starting point to develop more efficient tools to predict the membrane protein interaction site residues. PMID:23919118

  19. Modeling Protein Expression and Protein Signaling Pathways

    PubMed Central

    Telesca, Donatello; Müller, Peter; Kornblau, Steven M.; Suchard, Marc A.; Ji, Yuan

    2015-01-01

    High-throughput functional proteomic technologies provide a way to quantify the expression of proteins of interest. Statistical inference centers on identifying the activation state of proteins and their patterns of molecular interaction formalized as dependence structure. Inference on dependence structure is particularly important when proteins are selected because they are part of a common molecular pathway. In that case, inference on dependence structure reveals properties of the underlying pathway. We propose a probability model that represents molecular interactions at the level of hidden binary latent variables that can be interpreted as indicators for active versus inactive states of the proteins. The proposed approach exploits available expert knowledge about the target pathway to define an informative prior on the hidden conditional dependence structure. An important feature of this prior is that it provides an instrument to explicitly anchor the model space to a set of interactions of interest, favoring a local search approach to model determination. We apply our model to reverse-phase protein array data from a study on acute myeloid leukemia. Our inference identifies relevant subpathways in relation to the unfolding of the biological process under study. PMID:26246646

  20. Protein kinesis: The dynamics of protein trafficking and stability

    SciTech Connect

    1995-12-31

    The purpose of this conference is to provide a multidisciplinary forum for exchange of state-of-the-art information on protein kinesis. This volume contains abstracts of papers in the following areas: protein folding and modification in the endoplasmic reticulum; protein trafficking; protein translocation and folding; protein degradation; polarity; nuclear trafficking; membrane dynamics; and protein import into organelles.

  1. Protein flexibility as a biosignal.

    PubMed

    Zhao, Qinyi

    2010-01-01

    Dynamic properties of a protein are crucial for all protein functions, and those of signaling proteins are closely related to the biological function of living beings. The protein flexibility signal concept can be used to analyze this relationship. Protein flexibility controls the rate of protein conformational change and influences protein function. The modification of protein flexibility results in a change of protein activity. The logical nature of protein flexibility cannot be explained by applying the principles of protein three-dimensional structure theory or conformation concept. Signaling proteins show high protein flexibility. Many properties of signaling can be traced back to the dynamic natures of signaling protein. The action mechanism of volatile anesthetics and universal cellular reactions are related to flexibility in the change of signaling proteins. We conclude that protein dynamics is an enzyme-enhanced process, called dynamicase.

  2. Antimicrobial proteins: From old proteins, new tricks.

    PubMed

    Smith, Valerie J; Dyrynda, Elisabeth A

    2015-12-01

    This review describes the main types of antimicrobial peptides (AMPs) synthesised by crustaceans, primarily those identified in shrimp, crayfish, crab and lobster. It includes an overview of their range of microbicidal activities and the current landscape of our understanding of their gene expression patterns in different body tissues. It further summarises how their expression might change following various types of immune challenges. The review further considers proteins or protein fragments from crustaceans that have antimicrobial properties but are more usually associated with other biological functions, or are derived from such proteins. It discusses how these unconventional AMPs might be generated at, or delivered to, sites of infection and how they might contribute to crustacean host defence in vivo. It also highlights recent work that is starting to reveal the extent of multi-functionality displayed by some decapod AMPs, particularly their participation in other aspects of host protection. Examples of such activities include proteinase inhibition, phagocytosis, antiviral activity and haematopoiesis.

  3. Protein-protein Interactions using Radiolytic Footprinting

    SciTech Connect

    Takamoto,K.; Chance, M.

    2006-01-01

    Structural proteomics approaches using mass spectrometry are increasingly used in biology to examine the composition and structure of macromolecules. Hydroxyl radical-mediated protein footprinting using mass spectrometry has recently been developed to define structure, assembly, and conformational changes of macromolecules in solution based on measurements of reactivity of amino acid side chain groups with covalent modification reagents. Accurate measurements of side chain reactivity are achieved using quantitative liquid-chromatography-coupled mass spectrometry, whereas the side chain modification sites are identified using tandem mass spectrometry. In addition, the use of footprinting data in conjunction with computational modeling approaches is a powerful new method for testing and refining structural models of macromolecules and their complexes. In this review, we discuss the basic chemistry of hydroxyl radical reactions with peptides and proteins, highlight various approaches to map protein structure using radical oxidation methods, and describe state-of-the-art approaches to combine computational and footprinting data.

  4. Mechanisms Regulating Protein Localization.

    PubMed

    Bauer, Nicholas C; Doetsch, Paul W; Corbett, Anita H

    2015-10-01

    Cellular functions are dictated by protein content and activity. There are numerous strategies to regulate proteins varying from modulating gene expression to post-translational modifications. One commonly used mode of regulation in eukaryotes is targeted localization. By specifically redirecting the localization of a pool of existing protein, cells can achieve rapid changes in local protein function. Eukaryotic cells have evolved elegant targeting pathways to direct proteins to the appropriate cellular location or locations. Here, we provide a general overview of these localization pathways, with a focus on nuclear and mitochondrial transport, and present a survey of the evolutionarily conserved regulatory strategies identified thus far. We end with a description of several specific examples of proteins that exploit localization as an important mode of regulation.

  5. Mayaro virus proteins.

    PubMed

    Mezencio, J M; Rebello, M A

    1993-01-01

    Mayaro virus was grown in BHK-21 cells and purified by centrifugation in a potassium-tartrate gradient (5-50%). The electron microscopy analyses of the purified virus showed an homogeneous population of enveloped particles with 69 +/- 2.3 nm in diameter. Three structural virus proteins were identified and designated p1, p2 and p3. Their average molecular weight were p1, 54 KDa; p2, 50 KDa and p3, 34 KDa. In Mayaro virus infected Aedes albopictus cells and in BHK-21 infected cells we detected six viral proteins, in which three of them are the structural virus proteins and the other three were products from processing of precursors of viral proteins, whose molecular weights are 62 KDa, 64 KDa and 110 KDa. The 34 KDa protein was the first viral protein synthesized at 5 hours post-infection in both cell lines studied.

  6. TRIM proteins and diseases.

    PubMed

    Watanabe, Masashi; Hatakeyama, Shigetsugu

    2017-01-07

    Ubiquitination is one of the posttranslational modifications that regulates a number of intracellular events including signal transduction, protein quality control, transcription, cell cycle, apoptosis and development. The ubiquitin system functions as a garbage machine to degrade target proteins and as a regulator for several signalling pathways. Biochemical reaction of ubiquitination requires several enzymes including E1, E2 and E3, and E3 ubiquitin ligases play roles as receptors for recognizing target proteins. Most of the tripartite motif (TRIM) proteins are E3 ubiquitin ligases. Recent studies have shown that some TRIM proteins function as important regulators for a variety of diseases including cancer, inflammatory diseases, infectious diseases, neuropsychiatric disorders, chromosomal abnormalities and developmental diseases. In this review, we summarize the involvement of TRIM proteins in the aetiology of various diseases.

  7. Biofilm Matrix Proteins

    PubMed Central

    Fong, Jiunn N. C.; Yildiz, Fitnat H.

    2015-01-01

    Proteinaceous components of the biofilm matrix include secreted extracellular proteins, cell surface adhesins and protein subunits of cell appendages such as flagella and pili. Biofilm matrix proteins play diverse roles in biofilm formation and dissolution. They are involved in attaching cells to surfaces, stabilizing the biofilm matrix via interactions with exopolysaccharide and nucleic acid components, developing three-dimensional biofilm architectures, and dissolving biofilm matrix via enzymatic degradation of polysaccharides, proteins, and nucleic acids. In this chapter, we will review functions of matrix proteins in a selected set of microorganisms, studies of the matrix proteomes of Vibrio cholerae and Pseudomonas aeruginosa, and roles of outer membrane vesicles and of nucleoid-binding proteins in biofilm formation. PMID:26104709

  8. Protein oxidation and peroxidation

    PubMed Central

    Davies, Michael J.

    2016-01-01

    Proteins are major targets for radicals and two-electron oxidants in biological systems due to their abundance and high rate constants for reaction. With highly reactive radicals damage occurs at multiple side-chain and backbone sites. Less reactive species show greater selectivity with regard to the residues targeted and their spatial location. Modification can result in increased side-chain hydrophilicity, side-chain and backbone fragmentation, aggregation via covalent cross-linking or hydrophobic interactions, protein unfolding and altered conformation, altered interactions with biological partners and modified turnover. In the presence of O2, high yields of peroxyl radicals and peroxides (protein peroxidation) are formed; the latter account for up to 70% of the initial oxidant flux. Protein peroxides can oxidize both proteins and other targets. One-electron reduction results in additional radicals and chain reactions with alcohols and carbonyls as major products; the latter are commonly used markers of protein damage. Direct oxidation of cysteine (and less commonly) methionine residues is a major reaction; this is typically faster than with H2O2, and results in altered protein activity and function. Unlike H2O2, which is rapidly removed by protective enzymes, protein peroxides are only slowly removed, and catabolism is a major fate. Although turnover of modified proteins by proteasomal and lysosomal enzymes, and other proteases (e.g. mitochondrial Lon), can be efficient, protein hydroperoxides inhibit these pathways and this may contribute to the accumulation of modified proteins in cells. Available evidence supports an association between protein oxidation and multiple human pathologies, but whether this link is causal remains to be established. PMID:27026395

  9. Computer Models of Proteins

    NASA Technical Reports Server (NTRS)

    2000-01-01

    Dr. Marc Pusey (seated) and Dr. Craig Kundrot use computers to analyze x-ray maps and generate three-dimensional models of protein structures. With this information, scientists at Marshall Space Flight Center can learn how proteins are made and how they work. The computer screen depicts a proten structure as a ball-and-stick model. Other models depict the actual volume occupied by the atoms, or the ribbon-like structures that are crucial to a protein's function.

  10. Protein Crystal Quality Studies

    NASA Technical Reports Server (NTRS)

    1998-01-01

    Eddie Snell, Post-Doctoral Fellow the National Research Council (NRC) uses a reciprocal space mapping diffractometer for macromolecular crystal quality studies. The diffractometer is used in mapping the structure of macromolecules such as proteins to determine their structure and thus understand how they function with other proteins in the body. This is one of several analytical tools used on proteins crystallized on Earth and in space experiments. Photo credit: NASA/Marshall Space Flight Center (MSFC)

  11. Pressure cryocooling protein crystals

    DOEpatents

    Kim, Chae Un; Gruner, Sol M.

    2011-10-04

    Preparation of cryocooled protein crystal is provided by use of helium pressurizing and cryocooling to obtain cryocooled protein crystal allowing collection of high resolution data and by heavier noble gas (krypton or xenon) binding followed by helium pressurizing and cryocooling to obtain cryocooled protein crystal for collection of high resolution data and SAD phasing simultaneously. The helium pressurizing is carried out on crystal coated to prevent dehydration or on crystal grown in aqueous solution in a capillary.

  12. Chemical Synthesis of Proteins

    PubMed Central

    Nilsson, Bradley L.; Soellner, Matthew B.; Raines, Ronald T.

    2010-01-01

    Proteins have become accessible targets for chemical synthesis. The basic strategy is to use native chemical ligation, Staudinger ligation, or other orthogonal chemical reactions to couple synthetic peptides. The ligation reactions are compatible with a variety of solvents and proceed in solution or on a solid support. Chemical synthesis enables a level of control on protein composition that greatly exceeds that attainable with ribosome-mediated biosynthesis. Accordingly, the chemical synthesis of proteins is providing previously unattainable insight into the structure and function of proteins. PMID:15869385

  13. PIC: Protein Interactions Calculator

    PubMed Central

    Tina, K. G.; Bhadra, R.; Srinivasan, N.

    2007-01-01

    Interactions within a protein structure and interactions between proteins in an assembly are essential considerations in understanding molecular basis of stability and functions of proteins and their complexes. There are several weak and strong interactions that render stability to a protein structure or an assembly. Protein Interactions Calculator (PIC) is a server which, given the coordinate set of 3D structure of a protein or an assembly, computes various interactions such as disulphide bonds, interactions between hydrophobic residues, ionic interactions, hydrogen bonds, aromatic–aromatic interactions, aromatic–sulphur interactions and cation–π interactions within a protein or between proteins in a complex. Interactions are calculated on the basis of standard, published criteria. The identified interactions between residues can be visualized using a RasMol and Jmol interface. The advantage with PIC server is the easy availability of inter-residue interaction calculations in a single site. It also determines the accessible surface area and residue-depth, which is the distance of a residue from the surface of the protein. User can also recognize specific kind of interactions, such as apolar–apolar residue interactions or ionic interactions, that are formed between buried or exposed residues or near the surface or deep inside. PMID:17584791

  14. PIC: Protein Interactions Calculator.

    PubMed

    Tina, K G; Bhadra, R; Srinivasan, N

    2007-07-01

    Interactions within a protein structure and interactions between proteins in an assembly are essential considerations in understanding molecular basis of stability and functions of proteins and their complexes. There are several weak and strong interactions that render stability to a protein structure or an assembly. Protein Interactions Calculator (PIC) is a server which, given the coordinate set of 3D structure of a protein or an assembly, computes various interactions such as disulphide bonds, interactions between hydrophobic residues, ionic interactions, hydrogen bonds, aromatic-aromatic interactions, aromatic-sulphur interactions and cation-pi interactions within a protein or between proteins in a complex. Interactions are calculated on the basis of standard, published criteria. The identified interactions between residues can be visualized using a RasMol and Jmol interface. The advantage with PIC server is the easy availability of inter-residue interaction calculations in a single site. It also determines the accessible surface area and residue-depth, which is the distance of a residue from the surface of the protein. User can also recognize specific kind of interactions, such as apolar-apolar residue interactions or ionic interactions, that are formed between buried or exposed residues or near the surface or deep inside.

  15. Dietary proteins and angiogenesis.

    PubMed

    Medina, Miguel Ángel; Quesada, Ana R

    2014-01-17

    Both defective and persistent angiogenesis are linked to pathological situations in the adult. Compounds able to modulate angiogenesis have a potential value for the treatment of such pathologies. Several small molecules present in the diet have been shown to have modulatory effects on angiogenesis. This review presents the current state of knowledge on the potential modulatory roles of dietary proteins on angiogenesis. There is currently limited available information on the topic. Milk contains at least three proteins for which modulatory effects on angiogenesis have been previously demonstrated. On the other hand, there is some scarce information on the potential of dietary lectins, edible plant proteins and high protein diets to modulate angiogenesis.

  16. Consensus protein design

    PubMed Central

    Porebski, Benjamin T.; Buckle, Ashley M.

    2016-01-01

    A popular and successful strategy in semi-rational design of protein stability is the use of evolutionary information encapsulated in homologous protein sequences. Consensus design is based on the hypothesis that at a given position, the respective consensus amino acid contributes more than average to the stability of the protein than non-conserved amino acids. Here, we review the consensus design approach, its theoretical underpinnings, successes, limitations and challenges, as well as providing a detailed guide to its application in protein engineering. PMID:27274091

  17. Human Mitochondrial Protein Database

    National Institute of Standards and Technology Data Gateway

    SRD 131 Human Mitochondrial Protein Database (Web, free access)   The Human Mitochondrial Protein Database (HMPDb) provides comprehensive data on mitochondrial and human nuclear encoded proteins involved in mitochondrial biogenesis and function. This database consolidates information from SwissProt, LocusLink, Protein Data Bank (PDB), GenBank, Genome Database (GDB), Online Mendelian Inheritance in Man (OMIM), Human Mitochondrial Genome Database (mtDB), MITOMAP, Neuromuscular Disease Center and Human 2-D PAGE Databases. This database is intended as a tool not only to aid in studying the mitochondrion but in studying the associated diseases.

  18. TRIM proteins in development.

    PubMed

    Petrera, Francesca; Meroni, Germana

    2012-01-01

    TRIM proteins play important roles in several patho-physiological processes. Their common activity within the ubiquitylation pathway makes them amenable to a number of diverse biological roles. Many of the TRIM genes are highly and sometimes specifically expressed during embryogenesis, it is therefore not surprising that several of them might be involved in developmental processes. Here, we primarily discuss the developmental implications of two subgroups of TRIM proteins that conserved domain composition and functions from their invertebrate ancestors. The two groups are: the TRIM-NHL proteins implicated in miRNA processing regulation and the TRIM-FN3 proteins involved in ventral midline development.

  19. Engineering therapeutic protein disaggregases

    PubMed Central

    Shorter, James

    2016-01-01

    Therapeutic agents are urgently required to cure several common and fatal neurodegenerative disorders caused by protein misfolding and aggregation, including amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD), and Alzheimer’s disease (AD). Protein disaggregases that reverse protein misfolding and restore proteins to native structure, function, and localization could mitigate neurodegeneration by simultaneously reversing 1) any toxic gain of function of the misfolded form and 2) any loss of function due to misfolding. Potentiated variants of Hsp104, a hexameric AAA+ ATPase and protein disaggregase from yeast, have been engineered to robustly disaggregate misfolded proteins connected with ALS (e.g., TDP-43 and FUS) and PD (e.g., α-synuclein). However, Hsp104 has no metazoan homologue. Metazoa possess protein disaggregase systems distinct from Hsp104, including Hsp110, Hsp70, and Hsp40, as well as HtrA1, which might be harnessed to reverse deleterious protein misfolding. Nevertheless, vicissitudes of aging, environment, or genetics conspire to negate these disaggregase systems in neurodegenerative disease. Thus, engineering potentiated human protein disaggregases or isolating small-molecule enhancers of their activity could yield transformative therapeutics for ALS, PD, and AD. PMID:27255695

  20. Acanthamoeba castellanii STAT Protein

    PubMed Central

    Kicinska, Anna; Leluk, Jacek; Jarmuszkiewicz, Wieslawa

    2014-01-01

    STAT (signal transducers and activators of transcription) proteins are one of the important mediators of phosphotyrosine-regulated signaling in metazoan cells. We described the presence of STAT protein in a unicellular, free-living amoebae with a simple life cycle, Acanthamoeba castellanii. A. castellanii is the only, studied to date, Amoebozoan that does not belong to Mycetozoa but possesses STATs. A sequence of the A. castellanii STAT protein includes domains similar to those of the Dictyostelium STAT proteins: a coiled coil (characteristic for Dictyostelium STAT coiled coil), a STAT DNA-binding domain and a Src-homology domain. The search for protein sequences homologous to A. castellanii STAT revealed 17 additional sequences from lower eukaryotes. Interestingly, all of these sequences come from Amoebozoa organisms that belong to either Mycetozoa (slime molds) or Centramoebida. We showed that there are four separated clades within the slime mold STAT proteins. The A. castellanii STAT protein branches next to a group of STATc proteins from Mycetozoa. We also demonstrate that Amoebozoa form a distinct monophyletic lineage within the STAT protein world that is well separated from the other groups. PMID:25338074

  1. Protein intakes in India.

    PubMed

    Swaminathan, Sumathi; Vaz, Mario; Kurpad, Anura V

    2012-08-01

    Indian diets derive almost 60 % of their protein from cereals with relatively low digestibility and quality. There have been several surveys of diets and protein intakes in India by the National Nutrition Monitoring Board (NNMB) over the last 25 years, in urban and rural, as well as in slum dwellers and tribal populations. Data of disadvantaged populations from slums, tribals and sedentary rural Indian populations show that the protein intake (mainly from cereals) is about 1 gm/kg/day. However, the protein intake looks less promising in terms of the protein digestibility corrected amino acid score (PDCAAS), using lysine as the first limiting amino acid, where all populations, particularly rural and tribal, appear to have an inadequate quality to their protein intake. The protein: energy (PE) ratio is a measure of dietary quality, and has been used in the 2007 WHO/FAO/UNU report to define reference requirement values with which the adequacy of diets can be evaluated in terms of a protein quality corrected PE ratio. It is likely that about one third of this sedentary rural population is at risk of not meeting their requirements. These levels of risk of deficiency are in a population with relatively low BMI populations, whose diets are also inadequate in fruits and vegetables. Therefore, while the burden of enhancing the quality of protein intake in rural India exists, the quality of the diet, in general, represents a challenge that must be met.

  2. Self assembling proteins

    DOEpatents

    Yeates, Todd O.; Padilla, Jennifer; Colovos, Chris

    2004-06-29

    Novel fusion proteins capable of self-assembling into regular structures, as well as nucleic acids encoding the same, are provided. The subject fusion proteins comprise at least two oligomerization domains rigidly linked together, e.g. through an alpha helical linking group. Also provided are regular structures comprising a plurality of self-assembled fusion proteins of the subject invention, and methods for producing the same. The subject fusion proteins find use in the preparation of a variety of nanostructures, where such structures include: cages, shells, double-layer rings, two-dimensional layers, three-dimensional crystals, filaments, and tubes.

  3. Ultrafiltration of pegylated proteins

    NASA Astrophysics Data System (ADS)

    Molek, Jessica R.

    There is considerable clinical interest in the use of "second-generation" therapeutics produced by conjugation of a native protein with various polymers including polyethylene glycol (PEG). PEG--protein conjugates, so-called PEGylated proteins, can exhibit enhanced stability, half-life, and bioavailability. One of the challenges in the commercial production of PEGylated proteins is the purification required to remove unreacted polymer, native protein, and in many cases PEGylated proteins with nonoptimal degrees of conjugation. The overall objective of this thesis was to examine the use of ultrafiltration for the purification of PEGylated proteins. This included: (1) analysis of size-based separation of PEGylated proteins using conventional ultrafiltration membranes, (2) use of electrically-charged membranes to exploit differences in electrostatic interactions, and (3) examination of the effects of PEGylation on protein fouling. The experimental results were analyzed using appropriate theoretical models, with the underlying physical properties of the PEGylated proteins evaluated using size exclusion chromatography, capillary electrophoresis, dynamic light scattering, and reverse phase chromatography. PEGylated proteins were produced by covalent attachment of activated PEG to a protein via primary amines on the lysine residues. A simple model was developed for the reaction kinetics, which was used to explore the effect of reaction conditions and mode of operation on the distribution of PEGylated products. The effective size of the PEGylated proteins was evaluated using size exclusion chromatography, with appropriate correlations developed for the size in terms of the molecular weight of the native protein and attached PEG. The electrophoretic mobility of the PEGylated proteins were evaluated by capillary electrophoresis with the data in good agreement with a simple model accounting for the increase in protein size and the reduction in the number of protonated amine

  4. Regulation of protein secretion by ... protein secretion?

    PubMed

    Atmakuri, Krishnamohan; Fortune, Sarah M

    2008-09-11

    Mycobacterium tuberculosis (Mtb) requires an alternative protein secretion system, ESX1, for virulence. Recently, Raghavan et al. (2008) reported a new regulatory circuit that may explain how ESX1 activity is controlled during infection. Mtb appears to regulate ESX1 by modulating transcription of associated genes rather than structural components of the secretion system itself.

  5. Human Plasma Protein C

    PubMed Central

    Kisiel, Walter

    1979-01-01

    Protein C is a vitamin K-dependent protein, which exists in bovine plasma as a precursor of a serine protease. In this study, protein C was isolated to homogeneity from human plasma by barium citrate adsorption and elution, ammonium sulfate fractionation, DEAE-Sephadex chromatography, dextran sulfate agarose chromatography, and preparative polyacrylamide gel electrophoresis. Human protein C (Mr = 62,000) contains 23% carbohydrate and is composed of a light chain (Mr = 21,000) and a heavy chain (Mr = 41,000) held together by a disulfide bond(s). The light chain has an amino-terminal sequence of Ala-Asn-Ser-Phe-Leu- and the heavy chain has an aminoterminal sequence of Asp-Pro-Glu-Asp-Gln. The residues that are identical to bovine protein C are underlined. Incubation of human protein C with human α-thrombin at an enzyme to substrate weight ratio of 1:50 resulted in the formation of activated protein C, an enzyme with serine amidase activity. In the activation reaction, the apparent molecular weight of the heavy chain decreased from 41,000 to 40,000 as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. No apparent change in the molecular weight of the light chain was observed in the activation process. The heavy chain of human activated protein C also contains the active-site serine residue as evidenced by its ability to react with radiolabeled diisopropyl fluorophosphate. Human activated protein C markedly prolongs the kaolin-cephalin clotting time of human plasma, but not that of bovine plasma. The amidolytic and anticoagulant activities of human activated protein C were completely obviated by prior incubation of the enzyme with diisopropyl fluorophosphate. These results indicate that human protein C, like its bovine counterpart, exists in plasma as a zymogen and is converted to a serine protease by limited proteolysis with attendant anticoagulant activity. Images PMID:468991

  6. Engineered Protein Polymers

    DTIC Science & Technology

    2010-05-31

    of each pure polymer, we plan to combine the various polymer solutions in different ratios to tune the composition and physico-chemical properties...protein materials as vehicles for storage and delivery of small molecules. Each protein polymer under concentrations for particle formation ( vida

  7. Multidomain proteins under force.

    PubMed

    Valle-Orero, Jessica; Rivas-Pardo, Jaime Andrés; Popa, Ionel

    2017-04-28

    Advancements in single-molecule force spectroscopy techniques such as atomic force microscopy and magnetic tweezers allow investigation of how domain folding under force can play a physiological role. Combining these techniques with protein engineering and HaloTag covalent attachment, we investigate similarities and differences between four model proteins: I10 and I91-two immunoglobulin-like domains from the muscle protein titin, and two α + β fold proteins-ubiquitin and protein L. These proteins show a different mechanical response and have unique extensions under force. Remarkably, when normalized to their contour length, the size of the unfolding and refolding steps as a function of force reduces to a single master curve. This curve can be described using standard models of polymer elasticity, explaining the entropic nature of the measured steps. We further validate our measurements with a simple energy landscape model, which combines protein folding with polymer physics and accounts for the complex nature of tandem domains under force. This model can become a useful tool to help in deciphering the complexity of multidomain proteins operating under force.

  8. Archaeal chromatin proteins.

    PubMed

    Zhang, ZhenFeng; Guo, Li; Huang, Li

    2012-05-01

    Archaea, along with Bacteria and Eukarya, are the three domains of life. In all living cells, chromatin proteins serve a crucial role in maintaining the integrity of the structure and function of the genome. An array of small, abundant and basic DNA-binding proteins, considered candidates for chromatin proteins, has been isolated from the Euryarchaeota and the Crenarchaeota, the two major phyla in Archaea. While most euryarchaea encode proteins resembling eukaryotic histones, crenarchaea appear to synthesize a number of unique DNA-binding proteins likely involved in chromosomal organization. Several of these proteins (e.g., archaeal histones, Sac10b homologs, Sul7d, Cren7, CC1, etc.) have been extensively studied. However, whether they are chromatin proteins and how they function in vivo remain to be fully understood. Future investigation of archaeal chromatin proteins will lead to a better understanding of chromosomal organization and gene expression in Archaea and provide valuable information on the evolution of DNA packaging in cellular life.

  9. Protein Attachment on Nanodiamonds.

    PubMed

    Lin, Chung-Lun; Lin, Cheng-Huang; Chang, Huan-Cheng; Su, Meng-Chih

    2015-07-16

    A recent advance in nanotechnology is the scale-up production of small and nonaggregated diamond nanoparticles suitable for biological applications. Using detonation nanodiamonds (NDs) with an average diameter of ∼4 nm as the adsorbents, we have studied the static attachment of three proteins (myoglobin, bovine serum albumin, and insulin) onto the nanoparticles by optical spectroscopy, mass spectrometry, and dynamic light scattering, and electrophoretic zeta potential measurements. Results show that the protein surface coverage is predominantly determined by the competition between protein-protein and protein-ND interactions, giving each protein a unique and characteristic structural configuration in its own complex. Specifically, both myoglobin and bovine serum albumin show a Langmuir-type adsorption behavior, forming 1:1 complexes at saturation, whereas insulin folds into a tightly bound multimer before adsorption. The markedly different adsorption patterns appear to be independent of the protein concentration and are closely related to the affinity of the individual proteins for the NDs. The present study provides a fundamental understanding for the use of NDs as a platform for nanomedical drug delivery.

  10. Poxviral Ankyrin Proteins

    PubMed Central

    Herbert, Michael H.; Squire, Christopher J.; Mercer, Andrew A

    2015-01-01

    Multiple repeats of the ankyrin motif (ANK) are ubiquitous throughout the kingdoms of life but are absent from most viruses. The main exception to this is the poxvirus family, and specifically the chordopoxviruses, with ANK repeat proteins present in all but three species from separate genera. The poxviral ANK repeat proteins belong to distinct orthologue groups spread over different species, and align well with the phylogeny of their genera. This distribution throughout the chordopoxviruses indicates these proteins were present in an ancestral vertebrate poxvirus, and have since undergone numerous duplication events. Most poxviral ANK repeat proteins contain an unusual topology of multiple ANK motifs starting at the N-terminus with a C-terminal poxviral homologue of the cellular F-box enabling interaction with the cellular SCF ubiquitin ligase complex. The subtle variations between ANK repeat proteins of individual poxviruses suggest an array of different substrates may be bound by these protein-protein interaction domains and, via the F-box, potentially directed to cellular ubiquitination pathways and possible degradation. Known interaction partners of several of these proteins indicate that the NF-κB coordinated anti-viral response is a key target, whilst some poxviral ANK repeat domains also have an F-box independent affect on viral host-range. PMID:25690795

  11. Protein Kinases and Addiction

    PubMed Central

    Lee, Anna M.; Messing, Robert O.

    2011-01-01

    Although drugs of abuse have different chemical structures and interact with different protein targets, all appear to usurp common neuronal systems that regulate reward and motivation. Addiction is a complex disease that is thought to involve drug-induced changes in synaptic plasticity due to alterations in cell signaling, gene transcription, and protein synthesis. Recent evidence suggests that drugs of abuse interact with and change a common network of signaling pathways that include a subset of specific protein kinases. The best studied of these kinases are reviewed here and include extracellular signal-regulated kinase, cAMP-dependent protein kinase, cyclin-dependent protein kinase 5, protein kinase C, calcium/calmodulin-dependent protein kinase II, and Fyn tyrosine kinase. These kinases have been implicated in various aspects of drug addiction including acute drug effects, drug self-administration, withdrawal, reinforcement, sensitization, and tolerance. Identifying protein kinase substrates and signaling pathways that contribute to the addicted state may provide novel approaches for new pharma-cotherapies to treat drug addiction. PMID:18991950

  12. Sac phosphatase domain proteins.

    PubMed Central

    Hughes, W E; Cooke, F T; Parker, P J

    2000-01-01

    Advances in our understanding of the roles of phosphatidylinositol phosphates in controlling cellular functions such as endocytosis, exocytosis and the actin cytoskeleton have included new insights into the phosphatases that are responsible for the interconversion of these lipids. One of these is an entirely novel class of phosphatase domain found in a number of well characterized proteins. Proteins containing this Sac phosphatase domain include the yeast Saccharomyces cerevisiae proteins Sac1p and Fig4p. The Sac phosphatase domain is also found within the mammalian phosphoinositide 5-phosphatase synaptojanin and the yeast synaptojanin homologues Inp51p, Inp52p and Inp53p. These proteins therefore contain both Sac phosphatase and 5-phosphatase domains. This review describes the Sac phosphatase domain-containing proteins and their actions, with particular reference to the genetic and biochemical insights provided by study of the yeast Saccharomyces cerevisiae. PMID:10947947

  13. Proteins in unexpected locations.

    PubMed Central

    Smalheiser, N R

    1996-01-01

    Members of all classes of proteins--cytoskeletal components, secreted growth factors, glycolytic enzymes, kinases, transcription factors, chaperones, transmembrane proteins, and extracellular matrix proteins--have been identified in cellular compartments other than their conventional sites of action. Some of these proteins are expressed as distinct compartment-specific isoforms, have novel mechanisms for intercompartmental translocation, have distinct endogenous biological actions within each compartment, and are regulated in a compartment-specific manner as a function of physiologic state. The possibility that many, if not most, proteins have distinct roles in more than one cellular compartment has implications for the evolution of cell organization and may be important for understanding pathological conditions such as Alzheimer's disease and cancer. PMID:8862516

  14. Structures of membrane proteins

    PubMed Central

    Vinothkumar, Kutti R.; Henderson, Richard

    2010-01-01

    In reviewing the structures of membrane proteins determined up to the end of 2009, we present in words and pictures the most informative examples from each family. We group the structures together according to their function and architecture to provide an overview of the major principles and variations on the most common themes. The first structures, determined 20 years ago, were those of naturally abundant proteins with limited conformational variability, and each membrane protein structure determined was a major landmark. With the advent of complete genome sequences and efficient expression systems, there has been an explosion in the rate of membrane protein structure determination, with many classes represented. New structures are published every month and more than 150 unique membrane protein structures have been determined. This review analyses the reasons for this success, discusses the challenges that still lie ahead, and presents a concise summary of the key achievements with illustrated examples selected from each class. PMID:20667175

  15. Transdermal delivery of proteins.

    PubMed

    Kalluri, Haripriya; Banga, Ajay K

    2011-03-01

    Transdermal delivery of peptides and proteins avoids the disadvantages associated with the invasive parenteral route of administration and other alternative routes such as the pulmonary and nasal routes. Since proteins have a large size and are hydrophilic in nature, they cannot permeate passively across the skin due to the stratum corneum which allows the transport of only small lipophilic drug molecules. Enhancement techniques such as chemical enhancers, iontophoresis, microneedles, electroporation, sonophoresis, thermal ablation, laser ablation, radiofrequency ablation and noninvasive jet injectors aid in the delivery of proteins by overcoming the skin barrier in different ways. In this review, these enhancement techniques that can enable the transdermal delivery of proteins are discussed, including a discussion of mechanisms, sterility requirements, and commercial development of products. Combination of enhancement techniques may result in a synergistic effect allowing increased protein delivery and these are also discussed.

  16. Protein crystallization in microgravity.

    PubMed

    Aibara, S; Shibata, K; Morita, Y

    1997-12-01

    A space experiment involving protein crystallization was conducted in a microgravity environment using the space shuttle "Endeavour" of STS-47, on a 9-day mission from September 12th to 20th in 1992. The crystallization was carried out according to a batch method, and 5 proteins were selected as flight samples for crystallization. Two of these proteins: hen egg-white lysozyme and co-amino acid: pyruvate aminotransferase from Pseudomonas sp. F-126, were obtained as single crystals of good diffraction quality. Since 1992 we have carried out several space experiments for protein crystallization aboard space shuttles and the space station MIR. Our experimental results obtained mainly from hen egg-white lysozyme are described below, focusing on the effects of microgravity on protein crystal growth.

  17. Protein expression-yeast.

    PubMed

    Nielsen, Klaus H

    2014-01-01

    Yeast is an excellent system for the expression of recombinant eukaryotic proteins. Both endogenous and heterologous proteins can be overexpressed in yeast (Phan et al., 2001; Ton and Rao, 2004). Because yeast is easy to manipulate genetically, a strain can be optimized for the expression of a specific protein. Many eukaryotic proteins contain posttranslational modifications that can be performed in yeast but not in bacterial expression systems. In comparison with mammalian cell culture expression systems, growing yeast is both faster and less expensive, and large-scale cultures can be performed using fermentation. While several different yeast expression systems exist, this chapter focuses on the budding yeast Saccharomyces cerevisiae and will briefly describe some options to consider when selecting vectors and tags to be used for protein expression. Throughout this chapter, the expression and purification of yeast eIF3 is shown as an example alongside a general scheme outline.

  18. Protein Unfolding and Alzheimer's

    NASA Astrophysics Data System (ADS)

    Cheng, Kelvin

    2012-10-01

    Early interaction events of beta-amyloid (Aβ) proteins with neurons have been associated with the pathogenesis of Alzheimer's disease. Knowledge pertaining to the role of lipid molecules, particularly cholesterol, in modulating the single Aβ interactions with neurons at the atomic length and picosecond time resolutions, remains unclear. In our research, we have used atomistic molecular dynamics simulations to explore early molecular events including protein insertion kinetics, protein unfolding, and protein-induced membrane disruption of Aβ in lipid domains that mimic the nanoscopic raft and non-raft regions of the neural membrane. In this talk, I will summarize our current work on investigating the role of cholesterol in regulating the Aβ interaction events with membranes at the molecular level. I will also explain how our results will provide new insights into understanding the pathogenesis of Alzheimer's disease associated with the Aβ proteins.

  19. Junin virus structural proteins.

    PubMed Central

    De Martínez Segovia, Z M; De Mitri, M I

    1977-01-01

    Polyacrylamide gel electrophoresis of purified Junin virus revealed six distinct structural polypeptides, two major and four minor ones. Four of these polypeptides appeared to be covalently linked with carbohydrate. The molecular weights of the six proteins, estimated by coelectrophoresis with marker proteins, ranged from 25,000 to 91,000. One of the two major components (number 3) was identified as a nucleoprotein and had a molecular weight of 64,000. It was the most prominent protein and was nonglycosylated. The other major protein (number 5), with a molecular weight of 38,000, was a glucoprotein and a component of the viral envelope. The location on the virion of three additional glycopeptides with molecular weights of 91,000, 72,000, and 52,000, together with a protein with a molecular weight of 25,000, was not well defined. PMID:189088

  20. Manipulating and Visualizing Proteins

    SciTech Connect

    Simon, Horst D.

    2003-12-05

    ProteinShop Gives Researchers a Hands-On Tool for Manipulating, Visualizing Protein Structures. The Human Genome Project and other biological research efforts are creating an avalanche of new data about the chemical makeup and genetic codes of living organisms. But in order to make sense of this raw data, researchers need software tools which let them explore and model data in a more intuitive fashion. With this in mind, researchers at Lawrence Berkeley National Laboratory and the University of California, Davis, have developed ProteinShop, a visualization and modeling program which allows researchers to manipulate protein structures with pinpoint control, guided in large part by their own biological and experimental instincts. Biologists have spent the last half century trying to unravel the ''protein folding problem,'' which refers to the way chains of amino acids physically fold themselves into three-dimensional proteins. This final shape, which resembles a crumpled ribbon or piece of origami, is what determines how the protein functions and translates genetic information. Understanding and modeling this geometrically complex formation is no easy matter. ProteinShop takes a given sequence of amino acids and uses visualization guides to help generate predictions about the secondary structures, identifying alpha helices and flat beta strands, and the coil regions that bind them. Once secondary structures are in place, researchers can twist and turn these pre-configurations until they come up with a number of possible tertiary structure conformations. In turn, these are fed into a computationally intensive optimization procedure that tries to find the final, three-dimensional protein structure. Most importantly, ProteinShop allows users to add human knowledge and intuition to the protein structure prediction process, thus bypassing bad configurations that would otherwise be fruitless for optimization. This saves compute cycles and accelerates the entire process, so

  1. Protein disulfide engineering.

    PubMed

    Dombkowski, Alan A; Sultana, Kazi Zakia; Craig, Douglas B

    2014-01-21

    Improving the stability of proteins is an important goal in many biomedical and industrial applications. A logical approach is to emulate stabilizing molecular interactions found in nature. Disulfide bonds are covalent interactions that provide substantial stability to many proteins and conform to well-defined geometric conformations, thus making them appealing candidates in protein engineering efforts. Disulfide engineering is the directed design of novel disulfide bonds into target proteins. This important biotechnological tool has achieved considerable success in a wide range of applications, yet the rules that govern the stabilizing effects of disulfide bonds are not fully characterized. Contrary to expectations, many designed disulfide bonds have resulted in decreased stability of the modified protein. We review progress in disulfide engineering, with an emphasis on the issue of stability and computational methods that facilitate engineering efforts.

  2. Proteins, fluctuations and complexity

    SciTech Connect

    Frauenfelder, Hans; Chen, Guo; Fenimore, Paul W

    2008-01-01

    Glasses, supercooled liquids, and proteins share common properties, in particular the existence of two different types of fluctuations, {alpha} and {beta}. While the effect of the {alpha} fluctuations on proteins has been known for a few years, the effect of {beta} fluctuations has not been understood. By comparing neutron scattering data on the protein myoglobin with the {beta} fluctuations in the hydration shell measured by dielectric spectroscopy we show that the internal protein motions are slaved to these fluctuations. We also show that there is no 'dynamic transition' in proteins near 200 K. The rapid increase in the mean square displacement with temperature in many neutron scattering experiments is quantitatively predicted by the {beta} fluctuations in the hydration shell.

  3. [Controversies around diet proteins].

    PubMed

    Cichosz, Grazyna; Czeczot, Hanna

    2013-12-01

    Critical theories regarding proteins of anima origin are still and still popularized, though they are ungrounded from scientific point of view. Predominance of soya proteins over the animal ones in relation to their influence on calcium metabolism, bone break risk or risk of osteoporosis morbidity has not been confirmed in any honest, reliable research experiment. Statement, that sulphur amino acids influence disadvantageously on calcium metabolism of human organism and bone status, is completely groundless, the more so as presence of sulphur amino acids in diet (animal proteins are their best source) is the condition of endogenic synthesis of glutathione, the key antioxidant of the organism, and taurine stimulating brain functioning. Deficiency of proteins in the diet produce weakness of intellectual effectiveness and immune response. There is no doubt that limitation of consumption of animal proteins of standard value is not good for health.

  4. Drugging Membrane Protein Interactions

    PubMed Central

    Yin, Hang; Flynn, Aaron D.

    2016-01-01

    The majority of therapeutics target membrane proteins, accessible on the surface of cells, to alter cellular signaling. Cells use membrane proteins to transduce signals into cells, transport ions and molecules, bind the cell to a surface or substrate, and catalyze reactions. Newly devised technologies allow us to drug conventionally “undruggable” regions of membrane proteins, enabling modulation of protein–protein, protein–lipid, and protein–nucleic acid interactions. In this review, we survey the state of the art in high-throughput screening and rational design in drug discovery, and we evaluate the advances in biological understanding and technological capacity that will drive pharmacotherapy forward against unorthodox membrane protein targets. PMID:26863923

  5. Protein crystal growth

    NASA Technical Reports Server (NTRS)

    Bugg, Charles E.

    1993-01-01

    Proteins account for 50% or more of the dry weight of most living systems and play a crucial role in virtually all biological processes. Since the specific functions of essentially all biological molecules are determined by their three-dimensional structures, it is obvious that a detailed understanding of the structural makeup of a protein is essential to any systematic research pertaining to it. At the present time, protein crystallography has no substitute, it is the only technique available for elucidating the atomic arrangements within complicated biological molecules. Most macromolecules are extremely difficult to crystallize, and many otherwise exciting and promising projects have terminated at the crystal growth stage. There is a pressing need to better understand protein crystal growth, and to develop new techniques that can be used to enhance the size and quality of protein crystals. There are several aspects of microgravity that might be exploited to enhance protein crystal growth. The major factor that might be expected to alter crystal growth processes in space is the elimination of density-driven convective flow. Another factor that can be readily controlled in the absence of gravity is the sedimentation of growing crystal in a gravitational field. Another potential advantage of microgravity for protein crystal growth is the option of doing containerless crystal growth. One can readily understand why the microgravity environment established by Earth-orbiting vehicles is perceived to offer unique opportunities for the protein crystallographer. The near term objectives of the Protein Crystal Growth in a Microgravity Environment (PCG/ME) project is to continue to improve the techniques, procedures, and hardware systems used to grow protein crystals in Earth orbit.

  6. Regulation of protein turnover by heat shock proteins.

    PubMed

    Bozaykut, Perinur; Ozer, Nesrin Kartal; Karademir, Betul

    2014-12-01

    Protein turnover reflects the balance between synthesis and degradation of proteins, and it is a crucial process for the maintenance of the cellular protein pool. The folding of proteins, refolding of misfolded proteins, and also degradation of misfolded and damaged proteins are involved in the protein quality control (PQC) system. Correct protein folding and degradation are controlled by many different factors, one of the most important of which is the heat shock protein family. Heat shock proteins (HSPs) are in the class of molecular chaperones, which may prevent the inappropriate interaction of proteins and induce correct folding. On the other hand, these proteins play significant roles in the degradation pathways, including endoplasmic reticulum-associated degradation (ERAD), the ubiquitin-proteasome system, and autophagy. This review focuses on the emerging role of HSPs in the regulation of protein turnover; the effects of HSPs on the degradation machineries ERAD, autophagy, and proteasome; as well as the role of posttranslational modifications in the PQC system.

  7. Purifying protein complexes for mass spectrometry: applications to protein translation.

    PubMed

    Link, Andrew J; Fleischer, Tracey C; Weaver, Connie M; Gerbasi, Vincent R; Jennings, Jennifer L

    2005-03-01

    Proteins control and mediate most of the biological activities in the cell. In most cases, proteins either interact with regulatory proteins or function in large molecular assemblies to carryout biological processes. Understanding the functions of individual proteins requires the identification of these interacting proteins. With its speed and sensitivity, mass spectrometry has become the dominant method for identifying components of protein complexes. This article reviews and discusses various approaches to purify protein complexes and analyze the proteins using mass spectrometry. As examples, methods to isolate and analyze protein complexes responsible for the translation of messenger RNAs into polypeptides are described.

  8. Prediction of protein-protein interactions: unifying evolution and structure at protein interfaces.

    PubMed

    Tuncbag, Nurcan; Gursoy, Attila; Keskin, Ozlem

    2011-06-01

    The vast majority of the chores in the living cell involve protein-protein interactions. Providing details of protein interactions at the residue level and incorporating them into protein interaction networks are crucial toward the elucidation of a dynamic picture of cells. Despite the rapid increase in the number of structurally known protein complexes, we are still far away from a complete network. Given experimental limitations, computational modeling of protein interactions is a prerequisite to proceed on the way to complete structural networks. In this work, we focus on the question 'how do proteins interact?' rather than 'which proteins interact?' and we review structure-based protein-protein interaction prediction approaches. As a sample approach for modeling protein interactions, PRISM is detailed which combines structural similarity and evolutionary conservation in protein interfaces to infer structures of complexes in the protein interaction network. This will ultimately help us to understand the role of protein interfaces in predicting bound conformations.

  9. NMCP/LINC proteins

    PubMed Central

    Ciska, Malgorzata; Moreno Díaz de la Espina, Susana

    2013-01-01

    Lamins are the main components of the metazoan lamina, and while the organization of the nuclear lamina of metazoans and plants is similar, there are apparently no genes encoding lamins or most lamin-binding proteins in plants. Thus, the plant lamina is not lamin-based and the proteins that form this structure are still to be characterized. Members of the plant NMCP/LINC/CRWN protein family share the typical tripartite structure of lamins, although the 2 exhibit no sequence similarity. However, given the many similarities between NMCP/LINC/CRWN proteins and lamins (structural organization, position of conserved regions, sub-nuclear distribution, solubility, and pattern of expression), these proteins are good candidates to carry out the functions of lamins in plants. Moreover, functional analysis of NMCP/LINC mutants has revealed their involvement in maintaining nuclear size and shape, another activity fulfilled by lamins. This review summarizes the current understanding of NMCP/LINC proteins and discusses future studies that will be required to demonstrate definitively that these proteins are plant analogs of lamins. PMID:24128696

  10. TRIM proteins in cancer.

    PubMed

    Cambiaghi, Valeria; Giuliani, Virginia; Lombardi, Sara; Marinelli, Cristiano; Toffalorio, Francesca; Pelicci, Pier Giuseppe

    2012-01-01

    Some members of the tripartite motif (TRIM/RBCC) protein family are thought to be important regulators of carcinogenesis. This is not surprising as the TRIM proteins are involved in several biological processes, such as cell growth, development and cellular differentiation and alteration of these proteins can affect transcriptional regulation, cell proliferation and apoptosis. In particular, four TRIM family genes are frequently translocated to other genes, generating fusion proteins implicated in cancer initiation and progression. Among these the most famous is the promyelocytic leukaemia gene PML, which encodes the protein TRIM19. PML is involved in the t(15;17) translocation that specifically occurs in Acute Promyelocytic Leukaemia (APL), resulting in a PML-retinoic acid receptor-alpha (PML-RARalpha) fusion protein. Other members of the TRIM family are linked to cancer development without being involved in chromosomal re-arrangements, possibly through ubiquitination or loss of tumour suppression functions. This chapter discusses the biological functions of TRIM proteins in cancer.

  11. Protein based Block Copolymers

    PubMed Central

    Rabotyagova, Olena S.; Cebe, Peggy; Kaplan, David L.

    2011-01-01

    Advances in genetic engineering have led to the synthesis of protein-based block copolymers with control of chemistry and molecular weight, resulting in unique physical and biological properties. The benefits from incorporating peptide blocks into copolymer designs arise from the fundamental properties of proteins to adopt ordered conformations and to undergo self-assembly, providing control over structure formation at various length scales when compared to conventional block copolymers. This review covers the synthesis, structure, assembly, properties, and applications of protein-based block copolymers. PMID:21235251

  12. Piezoelectric allostery of protein

    NASA Astrophysics Data System (ADS)

    Ohnuki, Jun; Sato, Takato; Takano, Mitsunori

    2016-07-01

    Allostery is indispensable for a protein to work, where a locally applied stimulus is transmitted to a distant part of the molecule. While the allostery due to chemical stimuli such as ligand binding has long been studied, the growing interest in mechanobiology prompts the study of the mechanically stimulated allostery, the physical mechanism of which has not been established. By molecular dynamics simulation of a motor protein myosin, we found that a locally applied mechanical stimulus induces electrostatic potential change at distant regions, just like the piezoelectricity. This novel allosteric mechanism, "piezoelectric allostery", should be of particularly high value for mechanosensor/transducer proteins.

  13. Proteins : paradigms of complexity /

    SciTech Connect

    Frauenfelder, Hans,

    2001-01-01

    Proteins are the working machines of living systems. Directed by the DNA, of the order of a few hundred building blocks, selected from twenty different amino acids, are covalently linked into a linear polypeptide chain. In the proper environment, the chain folds into the working protein, often a globule of linear dimensions of a few nanometers. The biologist considers proteins units from which living systems are built. Many physical scientists look at them as systems in which the laws of complexity can be studied better than anywhere else. Some of the results of such studies will be sketched.

  14. Protein crystallography prescreen kit

    DOEpatents

    Segelke, Brent W.; Krupka, Heike I.; Rupp, Bernhard

    2005-07-12

    A kit for prescreening protein concentration for crystallization includes a multiplicity of vials, a multiplicity of pre-selected reagents, and a multiplicity of sample plates. The reagents and a corresponding multiplicity of samples of the protein in solutions of varying concentrations are placed on sample plates. The sample plates containing the reagents and samples are incubated. After incubation the sample plates are examined to determine which of the sample concentrations are too low and which the sample concentrations are too high. The sample concentrations that are optimal for protein crystallization are selected and used.

  15. Protein crystallography prescreen kit

    DOEpatents

    Segelke, Brent W.; Krupka, Heike I.; Rupp, Bernhard

    2007-10-02

    A kit for prescreening protein concentration for crystallization includes a multiplicity of vials, a multiplicity of pre-selected reagents, and a multiplicity of sample plates. The reagents and a corresponding multiplicity of samples of the protein in solutions of varying concentrations are placed on sample plates. The sample plates containing the reagents and samples are incubated. After incubation the sample plates are examined to determine which of the sample concentrations are too low and which the sample concentrations are too high. The sample concentrations that are optimal for protein crystallization are selected and used.

  16. Protein Crystal Malic Enzyme

    NASA Technical Reports Server (NTRS)

    1992-01-01

    Malic Enzyme is a target protein for drug design because it is a key protein in the life cycle of intestinal parasites. After 2 years of effort on Earth, investigators were unable to produce any crystals that were of high enough quality and for this reason the structure of this important protein could not be determined. Crystals obtained from one STS-50 were of superior quality allowing the structure to be determined. This is just one example why access to space is so vital for these studies. Principal Investigator is Larry DeLucas.

  17. Protein Crystal Quality Studies

    NASA Technical Reports Server (NTRS)

    1998-01-01

    Eddie Snell (standing), Post-Doctoral Fellow the National Research Council (NRC),and Marc Pusey of Marshall Space Flight Center (MSFC) use a reciprocal space mapping diffractometer for marcromolecular crystal quality studies. The diffractometer is used in mapping the structure of marcromolecules such as proteins to determine their structure and thus understand how they function with other proteins in the body. This is one of several analytical tools used on proteins crystalized on Earth and in space experiments. Photo credit: NASA/Marshall Space Flight Center (MSFC)

  18. Emerging fluorescent protein technologies.

    PubMed

    Enterina, Jhon Ralph; Wu, Lanshi; Campbell, Robert E

    2015-08-01

    Fluorescent proteins (FPs), such as the Aequorea jellyfish green FP (GFP), are firmly established as fundamental tools that enable a wide variety of biological studies. Specifically, FPs can serve as versatile genetically encoded markers for tracking proteins, organelles, or whole cells, and as the basis for construction of biosensors that can be used to visualize a growing array of biochemical events in cells and tissues. In this review we will focus on emerging applications of FPs that represent unprecedented new directions for the field. These emerging applications include new strategies for using FPs in biosensing applications, and innovative ways of using FPs to manipulate protein function or gene expression.

  19. Evolution of proteins.

    NASA Technical Reports Server (NTRS)

    Dayhoff, M. O.

    1971-01-01

    The amino acid sequences of proteins from living organisms are dealt with. The structure of proteins is first discussed; the variation in this structure from one biological group to another is illustrated by the first halves of the sequences of cytochrome c, and a phylogenetic tree is derived from the cytochrome c data. The relative geological times associated with the events of this tree are discussed. Errors which occur in the duplication of cells during the evolutionary process are examined. Particular attention is given to evolution of mutant proteins, globins, ferredoxin, and transfer ribonucleic acids (tRNA's). Finally, a general outline of biological evolution is presented.

  20. [Phosphorylation of tau protein].

    PubMed

    Uchida, T; Ishiguro, K

    1990-05-01

    In aged human brain and particularly in Alzheimer's disease brain, paired helical filaments (PHFs) accumulate in the neuronal cell. Recently, it has been found that the highly phosphorylated tau protein, one of the microtubule-associated proteins (MAPs), is a component of PHF. The authors attempted to clarify the mechanism underlying the accumulation of PHF from the following two aspects; 1) What is the mechanism of phosphorylation of tau protein? 2) Is the highly phosphorylated tau protein capable of forming PHFs? From rat or bovine microtubule proteins we partially purified and characterized a novel protein kinase that specifically phosphorylated tau and MAP2 among many proteins in the brain extract, and which formed a PHF epitope on the phosphorylated human tau. This enzyme was one of the protein serine/threonine kinases and was independent of known second messengers. The phosphorylation of tau by this enzyme was stimulated by tubulin under the condition of microtubule formation, suggesting that the phosphorylation of tau could occur concomitantly with microtubule formation in the brain. Since this kinase was usually bound to tau but not directly to tubulin, the enzyme was associated with microtubules through tau. From these properties related to tau, this kinase is designated as tau protein kinase. The tau that been phosphorylated with this kinase using [gamma-32P]ATP as a phosphate donor, was digested by endoprotinase Lys-C to produce three labeled fragments, K1, K2 and K3. These three fragments were sequenced and the phosphorylation sites on tau by this kinase were identified. The K2 fragment overlapped with the tau-1 site known to be one of the phosphorylation site in PHF. This result strengthens the possibility that tau protein phosphorylated by tau protein kinase is incorporated into PHF. Tubulin binding sites on tau were located between K1 and K3 fragments, while K2 fragment was located in the neighboring to N-terminus of K1. No phosphorylated sites were

  1. Teaching resources. Protein phosphatases.

    PubMed

    Salton, Stephen R

    2005-03-01

    This Teaching Resource provides lecture notes and slides for a class covering the structure and function of protein phosphatases and is part of the course "Cell Signaling Systems: A Course for Graduate Students." The lecture begins with a discussion of the importance of phosphatases in physiology, recognized by the award of a Nobel Prize in 1992, and then proceeds to describe the two types of protein phosphatases: serine/threonine and tyrosine phosphatases. The information covered includes the structure, regulation, and substrate specificity of protein phosphatases, with an emphasis on their importance in disease and clinical settings.

  2. Electrochromatographic separation of proteins

    NASA Technical Reports Server (NTRS)

    Basak, S. K.; Velayudhan, A.; Kohlmann, K.; Ladisch, M. R.; Mitchell, C. A. (Principal Investigator)

    1995-01-01

    We have developed a modified electrochromatography system which minimizes Joule heating at electric field strengths up to 125 V/cm. A non-linear equilibrium model is described which incorporates electrophoretic mobility, hydrodynamic flow velocity, and an electrically induced concentration polarization at the surface of the stationary phase. This model is able to provide useful estimates of protein retention time and velocity in a column packed with Sephadex gel and subjected to an electric field. A correlation of electrophoretic mobility of peptide and proteins with respect to their charge, molecular mass, and asymmetry enables the selection of solute target molecules for electrochromatographic separations. Good separation of protein mixtures have been obtained.

  3. (PCG) Protein Crystal Growth Canavalin

    NASA Technical Reports Server (NTRS)

    1989-01-01

    (PCG) Protein Crystal Growth Canavalin. The major storage protein of leguminous plants and a major source of dietary protein for humans and domestic animals. It is studied in efforts to enhance nutritional value of proteins through protein engineerings. It is isolated from Jack Bean because of it's potential as a nutritional substance. Principal Investigator on STS-26 was Alex McPherson.

  4. Plant protein glycosylation

    PubMed Central

    Strasser, Richard

    2016-01-01

    Protein glycosylation is an essential co- and post-translational modification of secretory and membrane proteins in all eukaryotes. The initial steps of N-glycosylation and N-glycan processing are highly conserved between plants, mammals and yeast. In contrast, late N-glycan maturation steps in the Golgi differ significantly in plants giving rise to complex N-glycans with β1,2-linked xylose, core α1,3-linked fucose and Lewis A-type structures. While the essential role of N-glycan modifications on distinct mammalian glycoproteins is already well documented, we have only begun to decipher the biological function of this ubiquitous protein modification in different plant species. In this review, I focus on the biosynthesis and function of different protein N-linked glycans in plants. Special emphasis is given on glycan-mediated quality control processes in the ER and on the biological role of characteristic complex N-glycan structures. PMID:26911286

  5. Protein Model Database

    SciTech Connect

    Fidelis, K; Adzhubej, A; Kryshtafovych, A; Daniluk, P

    2005-02-23

    The phenomenal success of the genome sequencing projects reveals the power of completeness in revolutionizing biological science. Currently it is possible to sequence entire organisms at a time, allowing for a systemic rather than fractional view of their organization and the various genome-encoded functions. There is an international plan to move towards a similar goal in the area of protein structure. This will not be achieved by experiment alone, but rather by a combination of efforts in crystallography, NMR spectroscopy, and computational modeling. Only a small fraction of structures are expected to be identified experimentally, the remainder to be modeled. Presently there is no organized infrastructure to critically evaluate and present these data to the biological community. The goal of the Protein Model Database project is to create such infrastructure, including (1) public database of theoretically derived protein structures; (2) reliable annotation of protein model quality, (3) novel structure analysis tools, and (4) access to the highest quality modeling techniques available.

  6. Protein Colloidal Aggregation Project

    NASA Technical Reports Server (NTRS)

    Oliva-Buisson, Yvette J. (Compiler)

    2014-01-01

    To investigate the pathways and kinetics of protein aggregation to allow accurate predictive modeling of the process and evaluation of potential inhibitors to prevalent diseases including cataract formation, chronic traumatic encephalopathy, Alzheimer's Disease, Parkinson's Disease and others.

  7. Fully automated protein purification

    PubMed Central

    Camper, DeMarco V.; Viola, Ronald E.

    2009-01-01

    Obtaining highly purified proteins is essential to begin investigating their functional and structural properties. The steps that are typically involved in purifying proteins can include an initial capture, intermediate purification, and a final polishing step. Completing these steps can take several days and require frequent attention to ensure success. Our goal was to design automated protocols that will allow the purification of proteins with minimal operator intervention. Separate methods have been produced and tested that automate the sample loading, column washing, sample elution and peak collection steps for ion-exchange, metal affinity, hydrophobic interaction and gel filtration chromatography. These individual methods are designed to be coupled and run sequentially in any order to achieve a flexible and fully automated protein purification protocol. PMID:19595984

  8. Protein fabrication automation

    PubMed Central

    Cox, J. Colin; Lape, Janel; Sayed, Mahmood A.; Hellinga, Homme W.

    2007-01-01

    Facile “writing” of DNA fragments that encode entire gene sequences potentially has widespread applications in biological analysis and engineering. Rapid writing of open reading frames (ORFs) for expressed proteins could transform protein engineering and production for protein design, synthetic biology, and structural analysis. Here we present a process, protein fabrication automation (PFA), which facilitates the rapid de novo construction of any desired ORF from oligonucleotides with low effort, high speed, and little human interaction. PFA comprises software for sequence design, data management, and the generation of instruction sets for liquid-handling robotics, a liquid-handling robot, a robust PCR scheme for gene assembly from synthetic oligonucleotides, and a genetic selection system to enrich correctly assembled full-length synthetic ORFs. The process is robust and scalable. PMID:17242375

  9. Interactive protein manipulation

    SciTech Connect

    SNCrivelli@lbl.gov

    2003-07-01

    We describe an interactive visualization and modeling program for the creation of protein structures ''from scratch''. The input to our program is an amino acid sequence -decoded from a gene- and a sequence of predicted secondary structure types for each amino acid-provided by external structure prediction programs. Our program can be used in the set-up phase of a protein structure prediction process; the structures created with it serve as input for a subsequent global internal energy minimization, or another method of protein structure prediction. Our program supports basic visualization methods for protein structures, interactive manipulation based on inverse kinematics, and visualization guides to aid a user in creating ''good'' initial structures.

  10. Recombinant Collagenlike Proteins

    NASA Technical Reports Server (NTRS)

    Fertala, Andzej

    2007-01-01

    A group of collagenlike recombinant proteins containing high densities of biologically active sites has been invented. The method used to express these proteins is similar to a method of expressing recombinant procollagens and collagens described in U. S. Patent 5,593,859, "Synthesis of human procollagens and collagens in recombinant DNA systems." Customized collagenous proteins are needed for biomedical applications. In particular, fibrillar collagens are attractive for production of matrices needed for tissue engineering and drug delivery. Prior to this invention, there was no way of producing customized collagenous proteins for these and other applications. Heretofore, collagenous proteins have been produced by use of such biological systems as yeasts, bacteria, and transgenic animals and plants. These products are normal collagens that can also be extracted from such sources as tendons, bones, and hides. These products cannot be made to consist only of biologically active, specific amino acid sequences that may be needed for specific applications. Prior to this invention, it had been established that fibrillar collagens consist of domains that are responsible for such processes as interaction with cells, binding of growth factors, and interaction with a number of structural proteins present in the extracellular matrix. A normal collagen consists of a sequence of domains that can be represented by a corresponding sequence of labels, e.g., D1D2D3D4. A collagenlike protein of the present invention contains regions of collagen II that contain multiples of a single domain (e.g., D1D1D1D1 or D4D4D4D4) chosen for its specific biological activity. By virtue of the multiplicity of the chosen domain, the density of sites having that specific biological activity is greater than it is in a normal collagen. A collagenlike protein according to this invention can thus be made to have properties that are necessary for tissue engineering.

  11. Occupational protein contact dermatitis.

    PubMed

    Barbaud, Annick; Poreaux, Claire; Penven, Emmanuelle; Waton, Julie

    2015-01-01

    Occupational contact dermatitis is generally caused by haptens but can also be induced by proteins causing mainly immunological contact urticaria (ICU); chronic hand eczema in the context of protein contact dermatitis (PCD). In a monocentric retrospective study, from our database, only 31 (0.41%) of patients with contact dermatitis had positive skin tests with proteins: 22 had occupational PCD, 3 had non-occupational PCD, 5 occupational ICU and 1 cook had a neutrophilic fixed food eruption (NFFE) due to fish. From these results and analysis of literature, the characteristics of PCD can be summarized as follows. It is a chronic eczematous dermatitis, possibly exacerbated by work, suggestive if associated with inflammatory perionyxix and immediate erythema with pruritis, to be investigated when the patient resumes work after a period of interruption. Prick tests with the suspected protein-containing material are essential, as patch tests have negative results. In case of multisensitisation revealed by prick tests, it is advisable to analyse IgE against recombinant allergens. A history of atopy, found in 56 to 68% of the patients, has to be checked for. Most of the cases are observed among food-handlers but PCD can also be due to non-edible plants, latex, hydrolysed proteins or animal proteins. Occupational exposure to proteins can thus lead to the development of ICU. Reflecting hypersensitivity to very low concentrations of allergens, investigating ICU therefore requires caution and prick tests should be performed with a diluted form of the causative protein-containing product. Causes are food, especially fruit peel, non-edible plants, cosmetic products, latex, animals.

  12. Chirality and protein biosynthesis.

    PubMed

    Banik, Sindrila Dutta; Nandi, Nilashis

    2013-01-01

    Chirality is present at all levels of structural hierarchy of protein and plays a significant role in protein biosynthesis. The macromolecules involved in protein biosynthesis such as aminoacyl tRNA synthetase and ribosome have chiral subunits. Despite the omnipresence of chirality in the biosynthetic pathway, its origin, role in current pathway, and importance is far from understood. In this review we first present an introduction to biochirality and its relevance to protein biosynthesis. Major propositions about the prebiotic origin of biomolecules are presented with particular reference to proteins and nucleic acids. The problem of the origin of homochirality is unresolved at present. The chiral discrimination by enzymes involved in protein synthesis is essential for keeping the life process going. However, questions remained pertaining to the mechanism of chiral discrimination and concomitant retention of biochirality. We discuss the experimental evidence which shows that it is virtually impossible to incorporate D-amino acids in protein structures in present biosynthetic pathways via any of the two major steps of protein synthesis, namely aminoacylation and peptide bond formation reactions. Molecular level explanations of the stringent chiral specificity in each step are extended based on computational analysis. A detailed account of the current state of understanding of the mechanism of chiral discrimination during aminoacylation in the active site of aminoacyl tRNA synthetase and peptide bond formation in ribosomal peptidyl transferase center is presented. Finally, it is pointed out that the understanding of the mechanism of retention of enantiopurity has implications in developing novel enzyme mimetic systems and biocatalysts and might be useful in chiral drug design.

  13. Protein Nitrogen Determination

    NASA Astrophysics Data System (ADS)

    Nielsen, S. Suzanne

    The protein content of foods can be determined by numerous methods. The Kjeldahl method and the nitrogen combustion (Dumas) method for protein analysis are based on nitrogen determination. Both methods are official for the purposes of nutrition labeling of foods. While the Kjeldahl method has been used widely for over a hundred years, the recent availability of automated instrumentation for the Dumas method in many cases is replacing use of the Kjeldahl method.

  14. Colorimetric protein assay techniques.

    PubMed

    Sapan, C V; Lundblad, R L; Price, N C

    1999-04-01

    There has been an increase in the number of colorimetric assay techniques for the determination of protein concentration over the past 20 years. This has resulted in a perceived increase in sensitivity and accuracy with the advent of new techniques. The present review considers these advances with emphasis on the potential use of such technologies in the assay of biopharmaceuticals. The techniques reviewed include Coomassie Blue G-250 dye binding (the Bradford assay), the Lowry assay, the bicinchoninic acid assay and the biuret assay. It is shown that each assay has advantages and disadvantages relative to sensitivity, ease of performance, acceptance in the literature, accuracy and reproducibility/coefficient of variation/laboratory-to-laboratory variation. A comparison of the use of several assays with the same sample population is presented. It is suggested that the most critical issue in the use of a chromogenic protein assay for the characterization of a biopharmaceutical is the selection of a standard for the calibration of the assay; it is crucial that the standard be representative of the sample. If it is not possible to match the standard with the sample from the perspective of protein composition, then it is preferable to use an assay that is not sensitive to the composition of the protein such as a micro-Kjeldahl technique, quantitative amino acid analysis or the biuret assay. In a complex mixture it might be inappropriate to focus on a general method of protein determination and much more informative to use specific methods relating to the protein(s) of particular interest, using either specific assays or antibody-based methods. The key point is that whatever method is adopted as the 'gold standard' for a given protein, this method needs to be used routinely for calibration.

  15. Protein conducting nanopores

    NASA Astrophysics Data System (ADS)

    Harsman, Anke; Krüger, Vivien; Bartsch, Philipp; Honigmann, Alf; Schmidt, Oliver; Rao, Sanjana; Meisinger, Christof; Wagner, Richard

    2010-11-01

    About 50% of the cellular proteins have to be transported into or across cellular membranes. This transport is an essential step in the protein biosynthesis. In eukaryotic cells secretory proteins are transported into the endoplasmic reticulum before they are transported in vesicles to the plasma membrane. Almost all proteins of the endosymbiotic organelles chloroplasts and mitochondria are synthesized on cytosolic ribosomes and posttranslationally imported. Genetic, biochemical and biophysical approaches led to rather detailed knowledge on the composition of the translocon-complexes which catalyze the membrane transport of the preproteins. Comprehensive concepts on the targeting and membrane transport of polypeptides emerged, however little detail on the molecular nature and mechanisms of the protein translocation channels comprising nanopores has been achieved. In this paper we will highlight recent developments of the diverse protein translocation systems and focus particularly on the common biophysical properties and functions of the protein conducting nanopores. We also provide a first analysis of the interaction between the genuine protein conducting nanopore Tom40SC as well as a mutant Tom40SC (\\mathrm {S}_{54} \\to E ) containing an additional negative charge at the channel vestibule and one of its native substrates, CoxIV, a mitochondrial targeting peptide. The polypeptide induced a voltage-dependent increase in the frequency of channel closure of Tom40SC corresponding to a voltage-dependent association rate, which was even more pronounced for the Tom40SC S54E mutant. The corresponding dwelltime reflecting association/transport of the peptide could be determined with \\bar {t}_{\\mathrm {off}} \\cong 1.1 ms for the wildtype, whereas the mutant Tom40SC S54E displayed a biphasic dwelltime distribution (\\bar {t}_{\\mathrm {off}}^1 \\cong 0.4 ms \\bar {t}_{\\mathrm {off}}^2 \\cong 4.6 ms).

  16. Fast protein folding kinetics

    PubMed Central

    Gelman, Hannah; Gruebele, Martin

    2014-01-01

    Fast folding proteins have been a major focus of computational and experimental study because they are accessible to both techniques: they are small and fast enough to be reasonably simulated with current computational power, but have dynamics slow enough to be observed with specially developed experimental techniques. This coupled study of fast folding proteins has provided insight into the mechanisms which allow some proteins to find their native conformation well less than 1 ms and has uncovered examples of theoretically predicted phenomena such as downhill folding. The study of fast folders also informs our understanding of even “slow” folding processes: fast folders are small, relatively simple protein domains and the principles that govern their folding also govern the folding of more complex systems. This review summarizes the major theoretical and experimental techniques used to study fast folding proteins and provides an overview of the major findings of fast folding research. Finally, we examine the themes that have emerged from studying fast folders and briefly summarize their application to protein folding in general as well as some work that is left to do. PMID:24641816

  17. Motor proteins 1: kinesins.

    PubMed

    Bloom, G S; Endow, S A

    1995-01-01

    Progress regarding the kinesins is now being made at a rapid and accelerating rate. The in vivo-functions, and biophysical and enzymatic properties of kinesin itself are being explored at ever increasing levels of detail. The kinesin-related proteins now number several dozen, and although more is known about primary structure than function for most of the proteins, this trend is already reversing. For example, knowledge about the kinesin-related protein, ncd, is expanding rapidly, and more is already known about its three-dimensional structure than is known for kinesin heavy chain. This volume presents a comprehensive review of the major published works on kinesin and kinesin-related proteins. Hopefully, this manuscript will complement other recent review articles [17, 20, 25, 37, 60-62, 67, 69, 75, 85-88, 231, 233, 238, 244, 269-271, 281, 282, 292] or books [49, 227, 293] that have focused on more selective aspects of the kinesin family, or have been aimed more generally at MT motor proteins. In line with the stated purpose of the Protein Profile series, annual updates of the review on the kinesins are planned for at least the next few years.

  18. Protein phosphorylation and photorespiration.

    PubMed

    Hodges, M; Jossier, M; Boex-Fontvieille, E; Tcherkez, G

    2013-07-01

    Photorespiration allows the recycling of carbon atoms of 2-phosphoglycolate produced by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) oxygenase activity, as well as the removal of potentially toxic metabolites. The photorespiratory pathway takes place in the light, encompasses four cellular compartments and interacts with several other metabolic pathways and functions. Therefore, the regulation of this cycle is probably of paramount importance to plant metabolism, however, our current knowledge is poor. To rapidly respond to changing conditions, proteins undergo a number of different post-translational modifications that include acetylation, methylation and ubiquitylation, but protein phosphorylation is probably the most common. The reversible covalent addition of a phosphate group to a specific amino acid residue allows the modulation of protein function, such as activity, subcellular localisation, capacity to interact with other proteins and stability. Recent data indicate that many photorespiratory enzymes can be phosphorylated, and thus it seems that the photorespiratory cycle is, in part, regulated by protein phosphorylation. In this review, the known phosphorylation sites of each Arabidopsis thaliana photorespiratory enzyme and several photorespiratory-associated proteins are described and discussed. A brief account of phosphoproteomic protocols is also given since the published data compiled in this review are the fruit of this approach.

  19. Disease specific protein corona

    NASA Astrophysics Data System (ADS)

    Rahman, M.; Mahmoudi, M.

    2015-03-01

    It is now well accepted that upon their entrance into the biological environments, the surface of nanomaterials would be covered by various biomacromolecules (e.g., proteins and lipids). The absorption of these biomolecules, so called `protein corona', onto the surface of (nano)biomaterials confers them a new `biological identity'. Although the formation of protein coronas on the surface of nanoparticles has been widely investigated, there are few reports on the effect of various diseases on the biological identity of nanoparticles. As the type of diseases may tremendously changes the composition of the protein source (e.g., human plasma/serum), one can expect that amount and composition of associated proteins in the corona composition may be varied, in disease type manner. Here, we show that corona coated silica and polystyrene nanoparticles (after interaction with in the plasma of the healthy individuals) could induce unfolding of fibrinogen, which promotes release of the inflammatory cytokines. However, no considerable releases of inflammatory cytokines were observed for corona coated graphene sheets. In contrast, the obtained corona coated silica and polystyrene nanoparticles from the hypofibrinogenemia patients could not induce inflammatory cytokine release where graphene sheets do. Therefore, one can expect that disease-specific protein coronas can provide a novel approach for applying nanomedicine to personalized medicine, improving diagnosis and treatment of different diseases tailored to the specific conditions and circumstances.

  20. Cotton and Protein Interactions

    SciTech Connect

    Goheen, Steven C.; Edwards, J. V.; Rayburn, Alfred R.; Gaither, Kari A.; Castro, Nathan J.

    2006-06-30

    The adsorbent properties of important wound fluid proteins and cotton cellulose are reviewed. This review focuses on the adsorption of albumin to cotton-based wound dressings and some chemically modified derivatives targeted for chronic wounds. Adsorption of elastase in the presence of albumin was examined as a model to understand the interactive properties of these wound fluid components with cotton fibers. In the chronic non-healing wound, elastase appears to be over-expressed, and it digests tissue and growth factors, interfering with the normal healing process. Albumin is the most prevalent protein in wound fluid, and in highly to moderately exudative wounds, it may bind significantly to the fibers of wound dressings. Thus, the relative binding properties of both elastase and albumin to wound dressing fibers are of interest in the design of more effective wound dressings. The present work examines the binding of albumin to two different derivatives of cotton, and quantifies the elastase binding to the same derivatives following exposure of albumin to the fiber surface. An HPLC adsorption technique was employed coupled with a colorimetric enzyme assay to quantify the relative binding properties of albumin and elastase to cotton. The results of wound protein binding are discussed in relation to the porosity and surface chemistry interactions of cotton and wound proteins. Studies are directed to understanding the implications of protein adsorption phenomena in terms of fiber-protein models that have implications for rationally designing dressings for chronic wounds.