RESUMO
S-layers commonly cover archaeal cell envelopes and are composed of proteins that self-assemble into a paracrystalline surface structure. Despite their detection in almost all archaea, there are few reports investigating the structural properties of these proteins, with no reports exploring this topic for halophilic S-layers. The objective of the present study was to investigate the secondary and tertiary organization of the Haloferax volcanii S-layer protein. Such investigations were performed using circular dichroism, fluorescence spectroscopy, dynamic light scattering and transmission electron microscopy. The protein secondary structure is centered on ß-sheets and is affected by environmental pH, with higher disorder in more alkaline conditions. The pH can also affect the protein's tertiary structure, with higher tryptophan side-chain exposure to the medium under the same conditions. The concentrations of Na, Mg and Ca ions in the environment also affect the protein structures, with small changes in α-helix and ß-sheet content, as well as changes in tryptophan side chain exposure. These changes in turn influence the protein's functional properties, with cell envelope preparations revealing striking differences when in different salt conditions. Thermal denaturation assays revealed that the protein is stable. It has been reported that the S-layer protein N-glycosylation process is affected by external factors and the present study indicates for the first time changes in the protein structure.
Assuntos
Haloferax volcanii/química , Temperatura Alta , Glicoproteínas de Membrana/química , Metais/química , Salinidade , Haloferax volcanii/metabolismo , Concentração de Íons de Hidrogênio , Glicoproteínas de Membrana/metabolismo , Metais/metabolismo , Desnaturação Proteica , Estrutura Secundária de ProteínaRESUMO
AIMS: To examine the antioxidant activity of Bacterioruberin (Bctr)-rich extracts isolated from a hyperpigmented, genetically modified Haloferax volcanii strain (HVLON3) and to investigate the effect on cold-sensitive ram sperm cells. METHODS AND RESULTS: The strain HVLON3 produces higher Bctr amounts than most haloarchaea (220 ± 13 mg g-1 DW). HVLON3-Bctr extract has higher antioxidant activity than ß-carotene (threefold) as evaluated using 2,2 diphenyl-1-picrylhydrazyl combined with Electron Paramagnetic Resonance analysis (EC50 4·5 × 10-5 mol l-1 vs 13·9 × 10-5 mol l-1 respectively). Different concentrations of HVLON3-Bctr extracts were assayed on ram sperm after freezing/thawing and physiologically relevant parameters were examined. Extracts containing 7 and 20 µmol l-1 Bctr significantly improved cell viability (P < 0·0001), total and progressive motility (P < 0·0001) and sperm velocities (P = 0·0172 for curvilinear velocity VCL, P = 0·0268 for average path velocity VAP and P = 0·0181 for straight line velocity VSL) and did not affect other parameters evaluated. CONCLUSIONS: HVLON3 is an excellent source of natural microbial C50 carotenoids with applicability in Biotechnology, Biomedical and Veterinary fields. HVLON3 Bctr extract improves the quality of cryopreserved ram sperm cells and could be applied to increase insemination yields. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides an insight on the bioactive properties of a bioproduct derived from haloarchaea (carotenoids) which are so far underexploited.
Assuntos
Antioxidantes/farmacologia , Carotenoides/farmacologia , Haloferax volcanii/química , Espermatozoides/efeitos dos fármacos , Animais , Antioxidantes/metabolismo , Carotenoides/metabolismo , Criopreservação , Haloferax volcanii/genética , Haloferax volcanii/metabolismo , Masculino , Ovinos , Motilidade dos Espermatozoides/efeitos dos fármacos , Espermatozoides/citologiaRESUMO
Rhomboids are conserved intramembrane serine proteases involved in cell signaling processes. Their role in prokaryotes is scarcely known and remains to be investigated in Archaea. We previously constructed a rhomboid homologue deletion mutant (ΔrhoII) in Haloferax volcanii, which showed reduced motility, increased novobiocin sensitivity, and an N- glycosylation defect. To address the impact of rhoII deletion on H. volcanii physiology, the proteomes of mutant and parental strains were compared by shotgun proteomics. A total of 1847 proteins were identified (45.8% of H. volcanii predicted proteome), from which 103 differed in amount. Additionally, the mutant strain evidenced 99 proteins with altered electrophoretic migration, which suggested differential post-translational processing/modification. Integral membrane proteins that evidenced variations in concentration, electrophoretic migration, or semitryptic cleavage in the mutant were considered as potential RhoII targets. These included a PrsW protease homologue (which was less stable in the mutant strain), a predicted halocyanin, and six integral membrane proteins potentially related to the mutant glycosylation (S-layer glycoprotein, Agl15) and cell adhesion/motility (flagellin1, HVO_1153, PilA1, and PibD) defects. This study investigated for the first time the impact of a rhomboid protease on the whole proteome of an organism.
Assuntos
Proteínas Arqueais/genética , Deleção de Genes , Regulação da Expressão Gênica em Archaea , Haloferax volcanii/genética , Processamento de Proteína Pós-Traducional , Proteoma/genética , Proteínas Arqueais/classificação , Proteínas Arqueais/isolamento & purificação , Proteínas Arqueais/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Adesão Celular , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Endopeptidases/deficiência , Endopeptidases/genética , Ontologia Genética , Glicosilação , Haloferax volcanii/química , Haloferax volcanii/metabolismo , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Metaloproteínas/genética , Metaloproteínas/metabolismo , Anotação de Sequência Molecular , Proteoma/classificação , Proteoma/isolamento & purificação , Proteoma/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Especificidade por SubstratoRESUMO
Proteins present in the archaeal cell envelope play key roles in a variety of processes necessary for survival in extreme environments. The haloarchaeon Haloferax volcanii is a good model for membrane proteomic studies because its genome sequence is known, it can be genetically manipulated, and a number of studies at the "omics" level have been performed in this organism. This work reports an easy strategy to improve the resolution of acidic membrane proteins from H. volcanii by 2DE. The method is based on the solubilization, delipidation, and salt removal from membrane proteins. Due to the abundance of the S-layer glycoprotein (SLG) in membrane protein extracts, other proteins from the envelope are consequently underrepresented. Thus, a protocol to reduce the amount of the SLG by EDTA treatment was applied and 11 cm narrow range pH (3.9-5.1) IPG strips were used to fractionate the remaining proteins. Using this method, horizontal streaking was substantially decreased and at least 75 defined spots (20% of the predicted membrane proteome within this pI/Mw range) were reproducibly detected. Two of these spots were identified as thermosome subunit 1 and NADH dehydrogenase from H. volcanii, confirming that proteins from the membrane fraction were enriched. Removal of the SLG from membrane protein extracts can be applied to increase protein load for 2DE as well as for other proteomic methods.