• Agitation effects on morphology and protein productive fractions of filamentous and pelleted growth forms of recombinant Aspergillus niger

      el-Enshasy, Hesham; Kleine, Joachim; Rinas, Ursula; Helmholtz zENTRUM FÜR iNFEKTIONSFORSCHUNG (2006-10)
    • Biomacromolecular interactions, assemblies and machines: a structural view.

      Heinz, Dirk W; Weiss, Manfred S; Wendt, K Ulrich (Wiley and Sons, 2006-01-01)
    • Biosynthesis of the repeating units of the exopolysaccharides amylovoran from Erwinia amylovora and stewartan from Pantoea stewartii

      Langlotz, Christine; Schollmeyer, Martin; Coplin, David L.; Nimtz, Manfred; Geider, Klaus; Max-Planck-Institute for Cell Biology, Ladenburg, Germany (2012-06-27)
    • Crystal structure of a non-discriminating glutamyl-tRNA synthetase.

      Schulze, Jörg O; Masoumi, Ava; Nickel, Daniel; Jahn, Martina; Jahn, Dieter; Schubert, Wolf-Dieter; Heinz, Dirk W; Division of Structural Biology, German Research Centre for Biotechnology (GBF), Mascheroder Weg 1, D-38124 Braunschweig, Germany. (2006-09-01)
      Error-free protein biosynthesis is dependent on the reliable charging of each tRNA with its cognate amino acid. Many bacteria, however, lack a glutaminyl-tRNA synthetase. In these organisms, tRNA(Gln) is initially mischarged with glutamate by a non-discriminating glutamyl-tRNA synthetase (ND-GluRS). This enzyme thus charges both tRNA(Glu) and tRNA(Gln) with glutamate. Discriminating GluRS (D-GluRS), found in some bacteria and all eukaryotes, exclusively generates Glu-tRNA(Glu). Here we present the first crystal structure of a non-discriminating GluRS from Thermosynechococcus elongatus (ND-GluRS(Tel)) in complex with glutamate at a resolution of 2.45 A. Structurally, the enzyme shares the overall architecture of the discriminating GluRS from Thermus thermophilus (D-GluRS(Tth)). We confirm experimentally that GluRS(Tel) is non-discriminating and present kinetic parameters for synthesis of Glu-tRNA(Glu) and of Glu-tRNA(Gln). Anticodons of tRNA(Glu) (34C/UUC36) and tRNA(Gln) (34C/UUG36) differ only in base 36. The pyrimidine base of C36 is specifically recognized in D-GluRS(Tth) by the residue Arg358. In ND-GluRS(Tel) this arginine residue is replaced by glycine (Gly366) presumably allowing both cytosine and the bulkier purine base G36 of tRNA(Gln) to be tolerated. Most other ND-GluRS share this structural feature, leading to relaxed substrate specificity.
    • Crystal structure of the conserved domain of the DC lysosomal associated membrane protein: implications for the lysosomal glycocalyx.

      Wilke, Sonja; Krausze, Joern; Büssow, Konrad; Department of Molecular Structural Biology, Helmholtz Centre for Infection Research, Inhoffenstr, 7, 38124 Braunschweig, Germany. konrad@buessow.com. (2012)
      ABSTRACT:
    • Crystal structure of the electron transfer complex rubredoxin rubredoxin reductase of Pseudomonas aeruginosa.

      Hagelueken, Gregor; Wiehlmann, Lutz; Adams, Thorsten M; Kolmar, Harald; Heinz, Dirk W; Tümmler, Burkhard; Schubert, Wolf-Dieter; Molecular Host-Pathogen Interactions, Division of Structural Biology, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, D-38124 Braunschweig, Germany. (2007-07-24)
      Crude oil spills represent a major ecological threat because of the chemical inertness of the constituent n-alkanes. The Gram-negative bacterium Pseudomonas aeruginosa is one of the few bacterial species able to metabolize such compounds. Three chromosomal genes, rubB, rubA1, and rubA2 coding for an NAD(P)H:rubredoxin reductase (RdxR) and two rubredoxins (Rdxs) are indispensable for this ability. They constitute an electron transport (ET) pathway that shuttles reducing equivalents from carbon metabolism to the membrane-bound alkane hydroxylases AlkB1 and AlkB2. The RdxR-Rdx system also is crucial as part of the oxidative stress response in archaea or anaerobic bacteria. The redox couple has been analyzed in detail as a model system for ET processes. We have solved the structure of RdxR of P. aeruginosa both alone and in complex with Rdx, without the need for cross-linking, and both structures were refined at 2.40- and 2.45-A resolution, respectively. RdxR consists of two cofactor-binding domains and a C-terminal domain essential for the specific recognition of Rdx. Only a small number of direct interactions govern mutual recognition of RdxR and Rdx, corroborating the transient nature of the complex. The shortest distance between the redox centers is observed to be 6.2 A.
    • Crystal structure of the heme d1 biosynthesis enzyme NirE in complex with its substrate reveals new insights into the catalytic mechanism of S-adenosyl-L-methionine-dependent uroporphyrinogen III methyltransferases.

      Storbeck, Sonja; Saha, Sayantan; Krausze, Joern; Klink, Björn U; Heinz, Dirk W; Layer, Gunhild; Institute of Microbiology, Technische Universität Braunschweig, 38106 Braunschweig, Germany. (2011-07-29)
      During the biosynthesis of heme d(1), the essential cofactor of cytochrome cd(1) nitrite reductase, the NirE protein catalyzes the methylation of uroporphyrinogen III to precorrin-2 using S-adenosyl-L-methionine (SAM) as the methyl group donor. The crystal structure of Pseudomonas aeruginosa NirE in complex with its substrate uroporphyrinogen III and the reaction by-product S-adenosyl-L-homocysteine (SAH) was solved to 2.0 Å resolution. This represents the first enzyme-substrate complex structure for a SAM-dependent uroporphyrinogen III methyltransferase. The large substrate binds on top of the SAH in a "puckered" conformation in which the two pyrrole rings facing each other point into the same direction either upward or downward. Three arginine residues, a histidine, and a methionine are involved in the coordination of uroporphyrinogen III. Through site-directed mutagenesis of the nirE gene and biochemical characterization of the corresponding NirE variants the amino acid residues Arg-111, Glu-114, and Arg-149 were identified to be involved in NirE catalysis. Based on our structural and biochemical findings, we propose a potential catalytic mechanism for NirE in which the methyl transfer reaction is initiated by an arginine catalyzed proton abstraction from the C-20 position of the substrate.
    • Discontinuous and continuous separation of the monomeric and dimeric forms of human bone morphogenetic protein-2 from renaturation batches.

      Gueorguieva, Ludmila; Vallejo, Luis Felipe; Rinas, Ursula; Seidel-Morgenstern, Andreas; Otto-von-Guericke-Universität Magdeburg, Institut für Verfahrenstechnik, PO Box 4120, D-39106 Magdeburg, Germany. (2006-12-01)
      Bone morphogenetic protein-2 (BMP-2) is one of the most interesting of the approximately 14 BMPs which belong to the transforming-growth-factor-beta (TGF-beta) superfamily. BMP-2 induces bone formation and thus plays an important role as a pharmaceutical protein. Recently, rhBMP-2 has been produced in form of inactive inclusion bodies in Escherichia coli. After solubilization and renaturation the biologically active dimeric form of rhBMP-2 can be generated. However, inactive monomers of BMP-2 are also formed during the renaturation process which must be separated from the active dimeric BMP-2. The purpose of this paper is to present: (a) results of an experimental study of a chromatographic separation of the monomeric and dimeric forms; and (b) a concept for a continuous counter-current simulated moving bed (SMB) process. The capacity of heparin as stationary phase was estimated for different salt concentrations in the mobile phase. A simulation study of a three-zone SMB process was performed applying a two step salt gradient. The results reveal the potential of the process for the purification of the dimeric BMP-2.
    • Evolutionary relationship between initial enzymes of tetrapyrrole biosynthesis.

      Schulze, Jörg O; Schubert, Wolf-Dieter; Moser, Jürgen; Jahn, Dieter; Heinz, Dirk W (2006-05-19)
      Glutamate-1-semialdehyde 2,1-aminomutase (GSAM) is the second enzyme in the C(5) pathway of tetrapyrrole biosynthesis found in most bacteria, in archaea and in plants. It catalyzes the transamination of glutamate-1-semialdehyde to 5-aminolevulinic acid (ALA) in a pyridoxal 5'-phosphate (PLP)-dependent manner. We present the crystal structure of GSAM from the thermophilic cyanobacterium Thermosynechococcus elongatus (GSAM(Tel)) in its PLP-bound form at 2.85A resolution. GSAM(Tel) is a symmetric homodimer, whereas GSAM from Synechococcus (GSAM(Syn)) has been described as asymmetric. The symmetry of GSAM(Tel) thus challenges the previously proposed negative cooperativity between monomers of this enzyme. Furthermore, GSAM(Tel) reveals an extensive flexible region at the interface of the proposed complex of GSAM with glutamyl-tRNA reductase (GluTR), the preceding enzyme in tetrapyrrole biosynthesis. Compared to GSAM(Syn), the monomers of GSAM(Tel) are rotated away from each other along the dimerization interface by 10 degrees . The associated flexibility of GSAM may be essential for complex formation with GluTR to occur. Unexpectedly, we find that GSAM is structurally related to 5-aminolevulinate synthase (ALAS), the ALA-producing enzyme in the Shemin pathway of alpha-proteobacteria and non-plant eukaryotes. This structural relationship applies also to the corresponding subfamilies of PLP-dependent enzymes. We thus propose that the CoA-subfamily (including ALAS) and the aminotransferase subfamily II (including GSAM) are evolutionarily closely related and that ALAS may thus have evolved from GSAM.
    • Expression of protein complexes using multiple Escherichia coli protein co-expression systems: a benchmarking study.

      Busso, Didier; Peleg, Yoav; Heidebrecht, Tatjana; Romier, Christophe; Jacobovitch, Yossi; Dantes, Ada; Salim, Loubna; Troesch, Edouard; Schuetz, Anja; Heinemann, Udo; et al. (2011-08)
      Escherichia coli (E. coli) remains the most commonly used host for recombinant protein expression. It is well known that a variety of experimental factors influence the protein production level as well as the solubility profile of over-expressed proteins. This becomes increasingly important for optimizing production of protein complexes using co-expression strategies. In this study, we focus on the effect of the choice of the expression vector system: by standardizing experimental factors including bacterial strain, cultivation temperature and growth medium composition, we compare the effectiveness of expression technologies used by the partners of the Structural Proteomics in Europe 2 (SPINE2-complexes) consortium. Four different protein complexes, including three binary and one ternary complex, all known to be produced in the soluble form in E. coli, are used as the benchmark targets. The respective genes were cloned by each partner into their preferred set of vectors. The resulting constructs were then used for comparative co-expression analysis done in parallel and under identical conditions at a single site. Our data show that multiple strategies can be applied for the expression of protein complexes in high yield. While there is no 'silver bullet' approach that was infallible even for this small test set, our observations are useful as a guideline to delineate co-expression strategies for particular protein complexes.
    • Gallic Esters of 4,5-Dinitrocatechol as Potential Building Blocks for Thermotropic Liquid Crystals

      JUDELE, Roxana; LASCHAT, SABINE; BARO, ANGELIKA; NIMTZ, MANFRED (Elsevier Ltd, 2006-10-09)
      A series of unsubstituted and 1,4-disubstituted gallic catecholates 1, 6 and 7 as possible candidates for wedge-shaped mesogens were prepared starting from the respective benzene derivatives 2a–c and gallic esters 5a–h. The mesomorphic properties were investigated by DSC. However, only the 4,5-dinitro derivatives 1d,f–h with C8H17 and C10H21 to C12H25 alkyl side chains displayed mesophases, as evaluated by fluidity and optical anisotropy.
    • Glutamate recognition and hydride transfer by Escherichia coli glutamyl-tRNA reductase.

      Lüer, Corinna; Schauer, Stefan; Virus, Simone; Schubert, Wolf-Dieter; Heinz, Dirk W; Moser, Jürgen; Jahn, Dieter; Institute of Microbiology, Technical University Braunschweig, Germany. (2007-09)
      The initial step of tetrapyrrole biosynthesis in Escherichia coli involves the NADPH-dependent reduction by glutamyl-tRNA reductase (GluTR) of tRNA-bound glutamate to glutamate-1-semialdehyde. We evaluated the contribution of the glutamate moiety of glutamyl-tRNA to substrate specificity in vitro using a range of substrates and enzyme variants. Unexpectedly, we found that tRNA(Glu) mischarged with glutamine was a substrate for purified recombinant GluTR. Similarly unexpectedly, the substitution of amino acid residues involved in glutamate side chain binding (S109A, T49V, R52K) or in stabilizing the arginine 52 glutamate interaction (glutamate 54 and histidine 99) did not abrogate enzyme activity. Replacing glutamine 116 and glutamate 114, involved in glutamate-enzyme interaction near the aminoacyl bond to tRNA(Glu), by leucine and lysine, respectively, however, did abolish reductase activity. We thus propose that the ester bond between glutamate and tRNA(Glu) represents the crucial determinant for substrate recognition by GluTR, whereas the necessity for product release by a 'back door' exit allows for a degree of structural variability in the recognition of the amino acid moiety. Analyzing the esterase activity, which occured in the absence of NADPH, of GluTR variants using the substrate 4-nitrophenyl acetate confirmed the crucial role of cysteine 50 for thioester formation. Finally, the GluTR variant Q116L was observed to lack reductase activity whereas esterase activity was retained. Structure-based molecular modeling indicated that glutamine 116 may be crucial in positioning the nicotinamide group of NADPH to allow for productive hydride transfer to the substrate. Our data thus provide new information about the distinct function of active site residues of GluTR from E. coli.
    • Human lung tissue explants reveal novel interactions during Legionella pneumophila infections.

      Jäger, Jens; Marwitz, Sebastian; Tiefenau, Jana; Rasch, Janine; Shevchuk, Olga; Kugler, Christian; Goldmann, Torsten; Steinert, Michael; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2014-01)
      Histological and clinical investigations describe late stages of Legionnaires' disease but cannot characterize early events of human infection. Cellular or rodent infection models lack the complexity of tissue or have nonhuman backgrounds. Therefore, we developed and applied a novel model for Legionella pneumophila infection comprising living human lung tissue. We stimulated lung explants with L. pneumophila strains and outer membrane vesicles (OMVs) to analyze tissue damage, bacterial replication, and localization as well as the transcriptional response of infected tissue. Interestingly, we found that extracellular adhesion of L. pneumophila to the entire alveolar lining precedes bacterial invasion and replication in recruited macrophages. In contrast, OMVs predominantly bound to alveolar macrophages. Specific damage to septa and epithelia increased over 48 h and was stronger in wild-type-infected and OMV-treated samples than in samples infected with the replication-deficient, type IVB secretion-deficient DotA(-) strain. Transcriptome analysis of lung tissue explants revealed a differential regulation of 2,499 genes after infection. The transcriptional response included the upregulation of uteroglobin and the downregulation of the macrophage receptor with collagenous structure (MARCO). Immunohistochemistry confirmed the downregulation of MARCO at sites of pathogen-induced tissue destruction. Neither host factor has ever been described in the context of L. pneumophila infections. This work demonstrates that the tissue explant model reproduces realistic features of Legionnaires' disease and reveals new functions for bacterial OMVs during infection. Our model allows us to characterize early steps of human infection which otherwise are not feasible for investigations.
    • Impact of biofilm matrix components on interaction of commensal Escherichia coli with the gastrointestinal cell line HT-29.

      Wang, X; Rochon, M; Lamprokostopoulou, A; Lünsdorf, H; Nimtz, M; Römling, U (2006-10-01)
      Commensal Escherichia coli form biofilms at body temperature by expressing the extracellular matrix components curli fimbriae and cellulose. The role of curli fimbriae and cellulose in the interaction of commensal E. coli with the intestinal epithelial cell line HT-29 was investigated. Expression of curli fimbriae by the typical commensal isolate E. coli TOB1 caused adherence and internalization of the bacteria and triggered IL-8 production in HT-29 cells. In particular, induction of IL-8 production was complex and involved curli-bound flagellin. While cellulose alone had no effect on the interaction of TOB1 with HT-29 cells, co-expression of cellulose with curli fimbriae decreased adherence to, internalization and IL-8 induction of HT-29 cells. Investigation of a panel of commensal isolates showed a partial correlation between expression of curli fimbriae and enhanced internalization and IL-8 production. In addition, a high immunostimulatory flagellin was identified. Thus, the consequences of expression of extracellular matrix components on commensal bacterial-host interactions are complex.
    • In situ multi-wavelength fluorescence spectroscopy as effective tool to simultaneously monitor spore germination, metabolic activity and quantitative protein production in recombinant Aspergillus niger fed-batch cultures.

      Ganzlin, Markus; Marose, Stefan; Lu, Xin; Hitzmann, Bernd; Scheper, Thomas; Rinas, Ursula; Helmholtz Centre for Infection Research (former German Research Centre for Biotechnology - GBF), Inhoffenstr. 7, 38124 Braunschweig, Germany. (2007-12-01)
      The production of a mutant green fluorescent protein (S65TGFP), controlled by the maltose inducible glucoamylase promoter, was followed in situ in fed-batch cultures of recombinant Aspergillus niger using multi-wavelength fluorescence spectroscopy. Disturbance of quantitative product analysis by interfering fluorescence signals was resolved by using a set of defined combinations of excitation and emission wavelengths (lambda(ex)/lambda(em)). This technique resulted in excellent linearity between on-line signal and off-line determined S65TGFP concentrations. Spore germination was detectable in situ by monitoring the back scattered light intensity. Moreover, flavin-like fluorophores were identified as the dominating fungal host fluorophores. The time-dependent intensity of this fluorophore, potentially fungal flavin-containing oxidoreductase(s), did not correlate with the biomass concentration but correlated well with the fungal metabolic activity (e.g. respiratory activity). Other fluorophores commonly found in microbial cultures such NADH, pyridoxine and the aromatic amino acids, tryptophan, phenylalanine and tyrosine did not contribute significantly to the culture fluorescence of A. niger. Thus, multi-wavelength fluorescence spectroscopy has proven to be an effective tool for simultaneous on-line monitoring of the most relevant process variables in fungal cultures, e.g. spore germination, metabolic activity, and quantitative product formation.
    • In situ structural analysis of the Yersinia enterocolitica injectisome

      Kudryashev, M.; Stenta, M.; Schmelz, S.; Amstutz, M.; Wiesand, U.; Castano-Diez, D.; Degiacomi, M. T.; Munnich, S.; Bleck, C. K.; Kowal, J.; et al. (2013-07-30)
    • In-depth analysis of the Aspergillus niger glucoamylase (glaA) promoter performance using high-throughput screening and controlled bioreactor cultivation techniques.

      Ganzlin, Markus; Rinas, Ursula; Helmholtz Centre for Infection Research, (Former German Research Centre for Biotechnology, GBF), Inhoffenstrasse 7, Braunschweig, Germany. (2008-06-30)
      An in-depth characterization of the Aspergillus niger glucoamylase (glaA) promoter performance was carried out on defined medium employing multi-well high-throughput screening as well as controlled batch and fed-batch bioreactor culture techniques with GFP as a fluorescent reporter protein. A variety of metabolizable carbon substrates and non-metabolizable analogs were screened with regard to their effect on the glaA expression system. The results clearly demonstrate that only starch and its hydrolytic products, including glucose, act as inducers. However, induction of the glaA expression system through the monosaccharide glucose is significantly lower compared to starch and the higher molecular weight starch degradation products. All other 26 carbon substrates tested do not induce, or even, as in the case of the easily metabolizable monosaccharide xylose, repress glaA-promoter controlled gene expression in the presence of the inducing disaccharide maltose with an increase of repression strength by increasing xylose concentrations. The complex effect of glucose on glaA-promoter controlled expression was also analyzed using non-metabolizable glucose analogs, namely 5-thio-glucose and 2-deoxyglucose, which were identified as novel and potent inducers of the glaA expression system. The results show that the induction strength depends on the inducer concentration with a maximum at defined concentrations and lower induction or even repression at concentrations above. Moreover, controlled fed-batch cultivations using a high maltose feed rate with concomitant extracellular accumulation of glucose resulted in lower levels of the reporter protein compared to cultures with a low-maltose feed rate without extracellular glucose accumulation, thus supporting the conclusion that increasing the glucose concentration beyond a critical point reduces the induction strength or may even cause repression. This way, the speed of polymer hydrolysis, glucose uptake and intracellular breakdown can be fine-tuned for optimal fungal growth and the metabolic burden for glucoamylase synthesis can be limited adequately in response to nutrient availability.
    • Inclusion body anatomy and functioning of chaperone-mediated in vivo inclusion body disassembly during high-level recombinant protein production in Escherichia coli.

      Rinas, Ursula; Hoffmann, Frank; Betiku, Eriola; Estapé, David; Marten, Sabine; Biochemical Engineering Division, GBF German Research Center for Biotechnology, Mascheroder Weg 1, D-38124 Braunschweig, Germany. URI@gbf.de (2007-01-01)
      During production in recombinant Escherichia coli, the human basic fibroblast growth factor (hFGF-2) partly aggregates into stable cytoplasmic inclusion bodies. These inclusion bodies additionally contain significant amounts of the heat-shock chaperone DnaK, and putative DnaK substrates such as the elongation factor Tu (ET-Tu) and the metabolic enzymes dihydrolipoamide dehydrogenase (LpdA), tryptophanase (TnaA), and d-tagatose-1,6-bisphosphate aldolase (GatY). Guanidinium hydrochloride induced disaggregation studies carried out in vitro on artificial aggregates generated through thermal aggregation of purified hFGF-2 revealed identical disaggregation profiles as hFGF-2 inclusion bodies indicating that the heterogenic composition of inclusion bodies did not influence the strength of interactions of hFGF-2 in aggregates formed in vivo as inclusion bodies compared to those generated in vitro from native and pure hFGF-2 through thermal aggregation. Compared to unfolding of native hFGF-2, higher concentrations of denaturant were required to dissolve hFGF-2 aggregates showing that more energy is required for disruption of interactions in both types of protein aggregates compared to the unfolding of the native protein. In vivo dissolution of hFGF-2 inclusion bodies was studied through coexpression of chaperones of the DnaK and GroEL family and ClpB and combinations thereof. None of the chaperone combinations was able to completely prevent the initial formation of inclusion bodies, but upon prolonged incubation mediated disaggregation of otherwise stable inclusion bodies. The GroEL system was particularly efficient in inclusion body dissolution but did not lead to a corresponding increase in soluble hFGF-2 rather was promoting the proteolysis of the recombinant growth factor. Coproduction of the disaggregating DnaK system and ClpB in conjunction with small amounts of the chaperonins GroELS was most efficient in disaggregation with concomitant formation of soluble hFGF-2. Thus, fine-balanced coproduction of chaperone combinations can play an important role in the production of soluble recombinant proteins with a high aggregation propensity not through prevention of aggregation but predominantly through their disaggregating properties.
    • Isolation of isomangiferin from honeybush (Cyclopia subternata) using high-speed counter-current chromatography and high-performance liquid chromatography.

      de Beer, Dalene; Jerz, Gerold; Joubert, Elizabeth; Wray, Victor; Winterhalter, Peter; ARC Infruitec-Nietvoorbij, Stellenbosch, South Africa. dbeerd@arc.agric.za (2009-05-08)
      Isomangiferin was isolated from Cyclopia subternata using a multi-step process including extraction, liquid-liquid partitioning, high-speed counter-current chromatography (HSCCC) and semi-preparative reversed-phase high-performance liquid chromatography (HPLC). Enrichment of phenolic compounds in a methanol extract of C. subternata leaves was conducted using liquid-liquid partitioning with ethyl acetate-methanol-water (1:1:2, v/v). The enriched fraction was further fractionated using HSCCC with a ternary solvent system consisting of tert-butyl methyl ether-n-butanol-acetonitrile-water (3:1:1:5, v/v). Isomangiferin was isolated by semi-preparative reversed-phase HPLC from a fraction containing mostly mangiferin and isomangiferin. The chemical structure of isomangiferin was confirmed by LC-high-resolution electrospray ionization MS, as well as one- and two-dimensional NMR spectroscopy.
    • Isolation, characterisation and molecular imaging of a high-molecular-weight insect biliprotein, a member of the hexameric arylphorin protein family.

      Kayser, Hartmut; Mann, Karlheinz; Machaidze, Gia; Nimtz, Manfred; Ringler, Philippe; Müller, Shirley A; Aebi, Ueli; Institut für Allgemeine Zoologie und Endokrinologie, Universität Ulm, Germany. hartmut.kayser@uni-ulm.de (2009-05-29)
      The abundant blue hemolymph protein of the last instar larvae of the moth Cerura vinula was purified and characterized by protein-analytical, spectroscopic and electron microscopic methods. Amino acid sequences obtained from a large number of cleavage peptides revealed a high level of similarity of the blue protein with arylphorins from a number of other moth species. In particular, there is a high abundance of the aromatic amino acids tyrosine and phenylalanine amounting to about 19% of total amino acids and a low content of methionine (0.8%) in the Cerura protein. The mass of the native protein complex was studied by size-exclusion chromatography, analytical ultracentrifugation, dynamic light scattering and scanning transmission electron microscopy and found to be around 500 kDa. Denaturating gel electrophoresis and mass spectrometry suggested the presence of two proteins with masses of about 85 kDa. The native Cerura protein is, therefore, a hexameric complex of two different subunits of similar size, as is known for arylphorins. The protein was further characterized as a weakly acidic (pI approximately 5.5) glycoprotein containing mannose, glucose and N-acetylglucosamine in an approximate ratio of 10:1:1. The structure proposed for the most abundant oligosaccharide of the Cerura arylphorin was the same as already identified in arylphorins from other moths. The intense blue colour of the Cerura protein is due to non-covalent association with a bilin of novel structure at an estimated protein subunit-to-ligand ratio of 3:1. Transmission electron microscopy of the biliprotein showed single particles of cylindrical shape measuring about 13 nm in diameter and 9 nm in height. A small fraction of particles of the same diameter but half the height was likely a trimeric arylphorin dissociation intermediate. Preliminary three-dimensional reconstruction based on averaged transmission electron microscopy projections of the individual particles revealed a double-trimeric structure for the hexameric Cerura biliprotein complex, suggesting it to be a dimer of trimers.