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1.
Virology ; 593: 110017, 2024 05.
Artigo em Inglês | MEDLINE | ID: mdl-38382161

RESUMO

Bacteriophage Mu is a temperate phage known to infect various species of Enterobacteria, playing a role in bacterial mutation induction and horizontal gene transfer. The phage possesses two types of tail fibers important for host recognition, which enable it to expand its range of hosts. The alternate tail fibers are formed through the action of genes 49-50 or 52-51, allowing the Mu phage to recognize different surfaces of host cells. In a previous study, we presented the X-ray crystal structure of the C-terminal lipopolysaccharide (LPS)-binding domain of gene product (gp) 49, one of the subunits comprising the Mu tail fiber. In this study, we have determined the structure of the alternative tail fiber subunit, gp52, and compared it with other tail fibers. The results revealed that Mu phage employs different structural motifs for two individual tail fibers for recognizing different hosts.


Assuntos
Bacteriófago mu , Bacteriófagos , Bacteriófago mu/química , Bacteriófago mu/genética , Bacteriófagos/genética , Proteínas da Cauda Viral/genética
2.
J Pept Sci ; 27(6): e3308, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33586251

RESUMO

Cherimolacylopeptide E (1) is a cyclic hexapeptide isolated from the seeds of Annona cherimola. Peptide 1 reportedly exhibits potent cytotoxicity against KB cells (IC50 0.017 µM). To confirm the structure and bioactivity of 1, we conducted a total synthesis of its proposed structure. The synthesis was accomplished via solid-phase peptide elongation and macrocyclization by employing Fmoc/OAll-protected amino acids on 2-Cl-trityl resin. NMR analysis revealed that synthetic 1 exists in two conformations in pyridine-d5 . As the spectroscopic data of the major conformer of synthetic 1 were consistent with those of natural 1, the structure of cherimolacyclopeptide E was confirmed to be 1. However, our synthetic 1 exhibited low cytotoxicity against KB cells (IC50 > 100 µM). In contrast to previously-reported findings, our synthetic 1 exhibited little antibacterial activity against Escherichia coli.


Assuntos
Annona/química , Peptídeos Cíclicos/farmacologia , Técnicas de Síntese em Fase Sólida , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Células KB , Conformação Molecular , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/química
3.
J Biochem ; 166(6): 529-535, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31504613

RESUMO

In the history of viral research, one of the important biological features of bacteriophage Mu is the ability to expand its host range. For extending the host range, the Mu phage encodes two alternate tail fibre genes. Classical amber mutation experiments and genome sequence analysis of Mu phage suggested that gene products (gp) of geneS (gpS = gp49) and gene S' (gpS' = gp52) are tail fibres and that gene products of geneU (gpU = gp50) and geneU' (gpU' = gp51) work for tail fibre assembly or tail fibre chaperones. Depending on the gene orientation, a pair of genes 49-50 or 52-51 is expressed for producing different tail fibres that enable Mu phage to recognize different host cell surface. Since several fibrous proteins including some phage tail fibres employ their specific chaperone to facilitate folding and prevent aggregation, we expected that gp50 or gp51 would be a specific chaperone for gp49 and gp52, respectively. However, heterologous overexpression results for gp49 or gp52 (tail fibre subunit) together with gp51 and gp50, respectively, were also effective in producing soluble Mu tail fibres. Moreover, we successfully purified non-native gp49-gp51 and gp52-gp50 complexes. These facts showed that gp50 and gp51 were fungible and functional for both gp49 and gp52 each other.


Assuntos
Bacteriófago mu/química , Chaperonas Moleculares/química , Sequência de Aminoácidos , Bacteriófago mu/genética , Bacteriófago mu/isolamento & purificação , Sítios de Ligação , Cristalização , Lipopolissacarídeos/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/isolamento & purificação , Alinhamento de Sequência
4.
J Oleo Sci ; 67(10): 1279-1289, 2018 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-30210077

RESUMO

Herein, to achieve individual and concomitant quantifications of phospholipid classes, an absolute quantification 31P NMR method using an internal standard was examined. Phospholipid standards and dietary foods were dispersed to prepare test solutions in an anionic surfactant (sodium cholate) solution containing EDTA, as a modification based on a reported method. Each phospholipid class showed a reproducible chemical shift at a near-neutral test solution pH of 6.90±0.04 and temperature of 30.0±0.1°C. The quantity of synthetic phosphatidylcholine measured using 31P NMR was consistent with that measured by 1H NMR using an internal standard. As the principal phospholipid class of soybean and egg yolk lecithin is phosphatidylcholine, the measurement conditions of 31P NMR (pulse interval time and number of scans) were optimized such that minor phospholipids, including lysophospholipids, also present in lecithin could be quantified simultaneously. Phospholipid classes in commercial polar lipid samples derived from porcine brain, yeast, and soybean were individually quantified using the above conditions. Using phosphoserine as the internal standard material allowed the absolute molar quantity of the phospholipid class to be precisely determined with traceability to the SI. The determined molar amounts of phospholipid classes were then translated to the weight amount by assuming that each phospholipid class contained two stearic acid molecules as the constituent fatty acid. The calculated total contents of each phospholipid class by 31P NMR were in good agreement with those obtained by molybdenum blue colorimetry. Furthermore, the quantitative values of the principal phospholipid classes in the polar lipid samples obtained by 31P NMR corresponded in a broad view, however, was more informative for the separation of individual phospholipid species rather than the quantitative 2D thin-layer chromatography.


Assuntos
Espectroscopia de Ressonância Magnética/métodos , Fosfolipídeos/análise , Fosfolipídeos/isolamento & purificação , Hidrogênio , Fosfolipídeos/classificação , Fosfolipídeos/normas , Fósforo
5.
Anal Sci ; 32(7): 729-34, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27396652

RESUMO

Okadaic acid (OA), a lipophilic shellfish toxin, was accurately quantified using quantitative nuclear magnetic resonance with internal standards for the development of an authentic reference standard. Pyridine and the residual proton in methanol-d4 were used as removable internal standards to limit any contamination. They were calibrated based on a maleic acid certified reference material. Thus, the concentration of OA was traceable to the SI units through accurate quantitative NMR with an internal reference substance. Signals from the protons on the oxygenated and unsaturated carbons of OA were used for quantification. A reasonable accuracy was obtained by integrating between the lower and upper (13)C satellite signal range when more than 4 mg of OA was used. The best-determined purity was 97.4% (0.16% RSD) when 20 mg of OA was used. Dinophysistoxin-1, a methylated analog of OA having an almost identical spectrum, was also quantified by using the same methodology.


Assuntos
Dinoflagellida/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Toxinas Marinhas/análise , Ácido Okadáico/análise , Piranos/análise , Frutos do Mar/análise , Calibragem , Padrões de Referência , Sensibilidade e Especificidade
6.
FEMS Microbiol Lett ; 314(1): 75-80, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21054503

RESUMO

Clostridial cellulosomes are cellulolytic complexes that are formed by highly specific interactions between one of the repeated cohesin modules present in the scaffolding protein and a dockerin module of the catalytic components. Although Clostridium thermocellum Xyn11A dockerin has a typical C. thermocellum dockerin sequence, in which two amino acid residues are species specifically conserved within the two segments of the dockerin modules, it can recognize Clostridium josui cohesin modules in a non-species-specific manner. The importance of these two conserved amino acids, which are part of the recognition site of the cohesin and dockerin interaction, was investigated by introducing mutations into the first and/or the second segments of the Xyn11A dockerin. Mutations in the first segment did not affect the interactions between dockerin and C. thermocellum and C. josui cohesins. However, mutations in the second segment prevented binding to cohesin proteins. A second round of mutations within the first segment re-established the affinity for both the C. thermocellum and the C. josui cohesins. However, this was not observed for a 'conventional' dockerin, Xyn10C. These results suggest that the combination of the first and second dockerin segments is important for the target recognition.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ciclo Celular/metabolismo , Celulase/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Clostridium thermocellum/metabolismo , Complexos Multienzimáticos/metabolismo , Mutação , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Celulase/química , Celulase/genética , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Clostridium/química , Clostridium/genética , Clostridium/metabolismo , Clostridium thermocellum/química , Clostridium thermocellum/genética , Dados de Sequência Molecular , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Ligação Proteica , Alinhamento de Sequência , Coesinas
7.
J Chromatogr B Analyt Technol Biomed Life Sci ; 878(3-4): 442-8, 2010 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-20061194

RESUMO

A new approach for the separation and inline characterization of lipopolysaccharide (LPS) related compounds has been developed. The separation was based on the difference in the number of charged phosphate and ethanolamine groups, as non-stoichiometric substituents, on the polysaccharide backbone, and was achieved with reverse phase ion-pairing chromatography (RPIP-HPLC). Tributylamine was used as an ion-pair reagent. In the conditions used in this study, tributylammonium then binds to the LPS related compounds through the negatively charged phosphate groups. This changes the hydrophobicity of the analytes at different positions and allows for separation based on both the number and position of the substituents on the analyte. The RPIP-HPLC was found to be effective for the separation of the O,N-deacylated derivative (deON) and polysaccharide portion (PS) from the LPS of Escherichia coli C strain. Post-column fluorescence derivatization (FLD), using sodium periodate and taurine, was used to detect the separated LPS related species. On the other hand, the separated species were also detected by direct infusion into the ESI-Q-MS using a volatile ammonium acetate buffer rather than the more traditional potassium phosphate buffer. The signal to noise ratio (S/N ratio) was low for the total ion chromatogram, however, high S/N ratios as well as good resolution were attained by selected ion monitoring (SIM) using m/z numbers corresponding to species with different numbers of non-stoichiometric substituents. Five species for deON and ten species for PS were clearly identified on the SIM chromatogram on the RPIP-HPLC/ESI-Q-MS. Accordingly, the present method allows for the effective separation and inline identification of the species corresponding to the diverse non-stoichiometric substitutions in LPS related compounds.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Cromatografia de Fase Reversa/métodos , Lipopolissacarídeos/isolamento & purificação , Espectrometria de Massas por Ionização por Electrospray/métodos , Acetilação , Ânions , Escherichia coli/química , Troca Iônica , Lipopolissacarídeos/análise , Lipopolissacarídeos/química
8.
Rapid Commun Mass Spectrom ; 24(1): 43-8, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19957294

RESUMO

The lipopolysaccharide (LPS) of enterobacteria frequently contains various numbers of charged non-stoichiometric substituents such as phosphate (P) and ethanolamine (EtN) groups and a third residue of 3-deoxy-D-manno-2-octulosonic acid (KDO) on the R-core polysaccharide backbone. These substituents can modify the biological activities of LPS including varying the stability of the outer membrane, tolerance to cationic antibiotics, pathogenicity, and sensitivity to enterobacteria bacteriophages. These diverse substituents can be clearly detected in degraded samples of LPS from E. coli C using electrospray ionization single quadrupole mass spectrometry (ESI-Q-MS) from a 0.1 mg/mL solution in a 50:50 mixture of methanol and 10 mM ammonium acetate (pH 6.8). The O-deacylated derivative showed multiple peaks of [M-3H](3-) ions which corresponded to species having up to eight phosphates, two ethanolamines, and an additional KDO on the backbone of Hex(5) Hep(3) KDO(2) GlcN(2) C14:0(3-OH)(2). The major components of the O,N-deacylated derivative were the species associated with four and five phosphates on Hex(5) Hep(3) KDO(2) GlcN(2). The polysaccharide portion of LPS also revealed species which corresponded to Hex(5) Hep(3) KDO associated with two to four phosphates and an ethanolamine. The present method was proved to be useful to investigate the structural diversity of enterobacterial LPS.


Assuntos
Escherichia coli/química , Lipopolissacarídeos/análise , Lipopolissacarídeos/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Sequência de Aminoácidos , Substituição de Aminoácidos , Dados de Sequência Molecular
9.
FEMS Microbiol Lett ; 300(2): 249-55, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19811541

RESUMO

Cellulosomes are cellulolytic complexes produced by anaerobic bacteria, and are composed of a scaffolding protein and several catalytic components. The complexes are formed by highly specific interactions of one of the reiterated cohesin modules of the scaffolding protein with a dockerin module of the catalytic components. The affinities of a dockerin module of Clostridium thermocellum CelJ (Cel9D-Cel44A) for several cohesin modules from C. thermocellum and Clostridium josui scaffolding proteins were quantitatively measured by surface plasmon resonance analysis. The recombinant CelJ dockerin-containing protein interacted with three recombinant C. josui cohesin proteins as well as recombinant C. thermocellum cohesin proteins beyond the so-called 'species specificity' of the dockerin and cohesin interactions. However, this protein did not recognize a second cohesin module from the C. josui scaffolding protein, suggesting that the catalytic components are not necessarily arranged randomly on a scaffolding protein in native cellulosomes.


Assuntos
Proteínas de Bactérias/metabolismo , Celulase/metabolismo , Clostridium thermocellum/enzimologia , Sequência de Aminoácidos , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Dados de Sequência Molecular , Filogenia , Ligação Proteica , Homologia de Sequência de Aminoácidos , Ressonância de Plasmônio de Superfície , Coesinas
10.
J Chromatogr B Analyt Technol Biomed Life Sci ; 877(14-15): 1537-42, 2009 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-19375984

RESUMO

The O,N-deacylated derivative (deON) and polysaccharide part (PS) from the lipopolysaccharide (LPS) of Escherichia coli C strain were separated by strongly basic anion-exchange chromatography (SAX) based on the differences in the number of charged phosphate and ethanolamine substituents. They were also successfully separated and characterized by capillary zone electrophoresis and subsequent ESI-ion trap-MS (CZE/ESI-IT-MS). The O-deacylated LPS (deO) presented as a broad peak in CZE/ESI-IT-MS. However, more than twelve species could be discriminated by an extracted ion electropherogram (EIE) and monitoring the species which have different numbers of phosphate and ethanolamine substituents on polysaccharide backbone.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Eletroforese Capilar/métodos , Escherichia coli/química , Lipopolissacarídeos/química , Espectrometria de Massas/métodos
11.
Blood Coagul Fibrinolysis ; 17(4): 317-22, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16651876

RESUMO

Defects of platelet adhesion, aggregation, secretion, or procoagulant activities can lead to bleeding diathesis of variable severity. We used recombinant activated factor VII (rFVIIa) in the treatment of uncontrolled epistaxis in a patient with a qualitative platelet disorder. We aimed to assess the efficacy of a single rFVIIa dose (100 microg/kg) in the control of mild and severe refractory epistaxis, and evaluate the influence of rFVIIa on markers of platelet adhesion and aggregation during a period of hematological stability (i.e. non-bleeding, no medication). The efficacy study showed mild episodes of epistaxis could be successfully managed using a single rFVIIa (100 microg/kg) dose; however, severe bleedings were not well controlled, and platelet transfusion was required to achieve hemostasis. Hematological investigations showed ADP-induced and collagen-induced platelet aggregation increases from 20 to 34% and 16 to 30%, respectively, following rFVIIa administration. There were no differences between pre-dose and post-dose concentrations of membrane glycoproteins. rFVIIa may therefore induce platelet aggregation by activating a glycoprotein-independent aggregation pathway. rFVIIa may have a role in managing mild bleeding episodes not controlled using conventional measures in patients with a qualitative platelet disorder. Further research is needed to determine the mechanism of action, efficacy, and safety of rFVIIa in this population.


Assuntos
Fator VIIa/administração & dosagem , Hemorragia/tratamento farmacológico , Trombastenia/tratamento farmacológico , Pré-Escolar , Feminino , Seguimentos , Hemostáticos/administração & dosagem , Humanos , Injeções Intravenosas , Agregação Plaquetária/efeitos dos fármacos , Contagem de Plaquetas , Proteínas Recombinantes/administração & dosagem , Trombastenia/diagnóstico , Resultado do Tratamento
12.
FEMS Microbiol Lett ; 251(2): 305-11, 2005 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-16143459

RESUMO

The contribution of the lipid part of lipopolysaccharide (LPS) to recognition by minor spike H protein of bacteriophage phiX174 was investigated by comparing the interactions of H protein with LPS and its deacylated derivatives. The fluorescence and circular dichroism (CD) spectra of H protein increased upon binding to intact LPS and a partially deacylated derivative. In contrast, completely deacylated derivatives showed lower affinities and almost no fluorescence or CD changes of H protein. These results demonstrate that the lipid part of LPS is responsible for the conformational change of minor spike H protein, which would function as a trigger for phage DNA ejection for infection of the host cell.


Assuntos
Bacteriófago phi X 174/química , Lipídeos/farmacologia , Lipopolissacarídeos/farmacologia , Estrutura Secundária de Proteína/efeitos dos fármacos , Proteínas Estruturais Virais/química , Sítios de Ligação , Dicroísmo Circular , Lipídeos/química
13.
J Biosci ; 30(4): 475-81, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16184009

RESUMO

Kinetics of fatty acid binding ability of glycated human serum albumin (HSA) were investigated by fluorescent displacement technique with 1-anilino-8-naphtharene sulphonic acid (ANS method), and photometric detection of nonesterified-fatty-acid (NEFA method). Changing of binding affinities of glycated HSA toward oleic acid, linoleic acid, lauric acid, and caproic acid, were not observed by the ANS method. However, decreases of bind-ing capacities after 55 days glycation were confirmed by the NEFA method in comparison to control HSA. The decrease in binding affinities was: oleic acid (84%), linoleic acid (84%), lauric acid (87%), and caproic acid (90%), respectively. The decreases were consistent with decrease of the intact lysine residues in glycated HSA. The present observation indicates that HSA promptly loses its binding ability to fatty acid as soon as the lysine residues at fatty acid binding sites are glycated.


Assuntos
Ácidos Graxos/metabolismo , Glucose/metabolismo , Albumina Sérica/metabolismo , Aminoácidos/genética , Glicosilação , Humanos , Cinética , Ácido Oleico/metabolismo , Análise de Sequência de Proteína , Albumina Sérica/genética
14.
Biosci Biotechnol Biochem ; 68(4): 924-6, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15118324

RESUMO

The interaction between the type-II dockerin domain of the scaffoldin protein CipA and the type-II cohesin domain of the outer layer protein SdbA is the fundamental mechanism for anchoring the cellulosome to the cell surface of Clostridium thermocellum. We constructed and purified a dockerin polypeptide and a cohesin polypeptide, and determined affinity constants of the interaction between them by the surface plasmon resonance method. The dissociation constant (K(D)) value was 1.8 x 10(-9) M, which is a little larger than that for the combination of a type-I dockerin and a type-I cohesin.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Celulossomas/química , Celulossomas/metabolismo , Clostridium/química , Proteínas Nucleares/química , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , Clostridium/metabolismo , Proteínas Fúngicas , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Ressonância de Plasmônio de Superfície , Coesinas
15.
J Biol Chem ; 279(11): 9867-74, 2004 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-14688277

RESUMO

The cellulosome components are assembled into the cellulosome complex by the interaction between one of the repeated cohesin domains of a scaffolding protein and the dockerin domain of an enzyme component. We prepared five recombinant cohesin polypeptides of the Clostridium thermocellum scaffolding protein CipA, two dockerin polypeptides of C. thermocellum Xyn11A and Xyn10C, four cohesin polypeptides of Clostridium josui CipA, and two dockerin polypeptides of C. josui Aga27A and Cel8A, and qualitatively and quantitatively examined the cohesin-dockerin interactions within C. thermocellum and C. josui, respectively, and the species specificity of the cohesin-dockerin interactions between these two bacteria. Surface plasmon resonance (SPR) analysis indicated that there was a certain selectivity, with a maximal 34-fold difference in the K(D) values, in the cohesin-dockerin interactions within a combination of C. josui, although this was not detected by qualitative analysis. Affinity blotting analysis suggested that there was at least one exception to the species specificity in the cohesin-dockerin interactions, although species specificity was generally conserved among the cohesin and dockerin polypeptides from C. thermocellum and C. josui, i.e. the dockerin polypeptides of C. thermocellum Xyn11A exceptionally bound to the cohesin polypeptides from C. josui CipA. SPR analysis confirmed this exceptional binding. We discuss the relationship between the species specificity of the cohesin-dockerin binding and the conserved amino acid residues in the dockerin domains.


Assuntos
Proteínas de Bactérias/química , Celulase/química , Clostridium/metabolismo , Proteínas de Membrana/química , Proteínas Nucleares/química , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Proteínas de Ciclo Celular , Celulase/metabolismo , Proteínas Cromossômicas não Histona , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Proteínas Fúngicas , Cinética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Peptídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Especificidade por Substrato , Ressonância de Plasmônio de Superfície , Fatores de Tempo , Coesinas
16.
FEMS Microbiol Lett ; 226(2): 221-7, 2003 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-14553915

RESUMO

The binding of spike H and G proteins of bacteriophage phiX174 with lipopolysaccharides (LPSs) were evaluated by a competitive enzyme-linked plate assay using the biotin-labeled LPS of Escherichia coli C, one of a host strain, and the non-labeled LPSs having different R-core polysaccharide lengths. H protein promptly decreased its affinity when some saccharide residues were truncated from the outer R-core. However, G protein showed significant affinity to the LPSs lacking all the residues of the outer R-core and some of the inner R-core. Thus, G protein rather than H protein well recognized the residues of the inner R-core of LPS.


Assuntos
Bacteriófago phi X 174/fisiologia , Lipopolissacarídeos/química , Lipopolissacarídeos/metabolismo , Proteínas Estruturais Virais/metabolismo , Bacteriófago phi X 174/genética , Bacteriófago phi X 174/crescimento & desenvolvimento , Ligação Competitiva , Biotina/metabolismo , Sequência de Carboidratos , Escherichia coli/química , Lipopolissacarídeos/isolamento & purificação , Dados de Sequência Molecular , Ligação Proteica , Receptores Virais/química , Receptores Virais/isolamento & purificação , Receptores Virais/metabolismo , Salmonella typhimurium/química , Proteínas Estruturais Virais/isolamento & purificação
17.
Biosci Biotechnol Biochem ; 67(4): 869-76, 2003 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12784630

RESUMO

A histidine-tagged G protein of bacteriophage phi X174 (HisG) bound strongly with lipopolysaccharide (LPS) of Escherichia coli C, one of a phi X174-sensitive Ra strain. The dissociation constant, Kd, was measured to be 0.16 +/- 0.04 microM by fluorometric titration. HisG showed slightly less affinity to LPSs of the insensitive Rc and Rd 2 strains having shorter R-core polysaccharide sequences than that of the sensitive Ra strains. The difference between the two types of LPS was demonstrated by CD spectra; LPSs of the sensitive strains increased the signal intensity for beta-sheet, while the insensitive strains decreased it. The chemically degraded LPS derivatives lacking a hydrophobic lipid region showed much less affinity to HisG, indicating the importance of the lipid region of LPS for strong binding with HisG. On the other hand, since the degraded derivatives increased the intensity of CD spectra, the polysaccharide region is thought to contribute to the conformation change of the protein.


Assuntos
Bacteriófagos/química , Lipopolissacarídeos/metabolismo , Proteínas Estruturais Virais/metabolismo , Sítios de Ligação , Dicroísmo Circular , Escherichia coli/química , Fluorometria , Lipopolissacarídeos/química , Lipopolissacarídeos/farmacologia , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Relação Estrutura-Atividade , Proteínas Estruturais Virais/química
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