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Type-3 copper proteins (T3CPs) are complex proteins which share similar active sites. Two copper atoms (CuA and CuB) bind dioxygen as a peroxide in a side on coordination. This protein family comprises the enzymes tyrosinase and catechol oxidase as well as the oxygen transporter hemocyanin. T3CPs occur in almost all organisms and exhibit a number of essential functions. In particular, they are involved in all kinds of enzymatic browning reactions and immune defense. The chemical basis of the two catalytic processes, i.e., the o-hydroxylation of monophenols and the two-electron oxidation to o-quinones, is still discussed. Investigations on natural enzymes with known crystal structures, along with site directed mutagenesis experiments, provide deeper insight into mechanistic details of these reactions. In particular, they shed light on the deprotonation of monophenolic substrates which appears to be required for the enzymatic conversion to o-quinones. Moreover, they allow drawing definite conclusions regarding the role of particular amino acids (glutamate and asparagine/aspartate) in these processes, which in turn provides a structure-based argument for the functional difference between tyrosinases and catechol oxidases.

Grapevine is one of the most important fruit plants throughout the world. Sequencing of the grape genome in 2007 enabled in-depth analyses of the grape proteome. Whereas many studies addressed changes in proteomic composition of grapes during ripening, we focused on the proteome of mature grape berries from Dornfelder, a characteristic red wine grape for Germany. Current data-independent acquisition proteomics technology enables the analysis of proteomic compositions in a degree of accuracy that was unreachable only a few years ago. Using a label-free proteomics approach, we quantified 712 proteins in mature Dornfelder grape berries, of which 650 could be annotated by the Blast2GO software. Besides identification of proteins, our analysis provides protein amounts using the TOP3 absolute quantification approach. Most of the proteins (200) in mature Dornfelder grape berries are involved in stress response. In addition, all glycolytic key enzymes were detected in mature grape berries suggesting that glycolysis is still active, whereas sugar accumulation through gluconeogenesis utilizing malate as substrate seems to play a minor role.

Hemocyanins are respiratory dioxygen carrier proteins found in many arthropods including ancient terrestrial species such as spiders and scorpions as well as marine horseshoe crabs. As hemocyanins are highly conserved in this lineage, it is possible to observe an evolutionary descent through its subunits and their overall structure. Unfortunately, little is known about the structure and function of hexapod hemocyanins. Using recent springtail taxa (Collembola) as models for basal hexapods, and the help of electron microscopy, light scattering, SDS PAGE, and Western blot, we could demonstrate for the first time the presence of 2 × 6-meric hemocyanins in the hemolymph of hexapods. The quaternary structure is composed of at least two different subunits and looks nearly identical to the hemocyanin found in decapod crustaceans. In addition, homology modeling and western blotting suggest a close structural relationship between collembolan and crustacean hemocyanin. Such a respiratory protein was possibly helpful in the early terrestrialization process of ancient Collembola. In addition, physiological adaptations to hypoxic or temporarily anoxic conditions could be a possible explanation for the presence of this respiratory protein. Nevertheless, it has to be concluded that the primary benefit of hemocyanin for springtails remains unclear.

Bentonite is the only fining agent which can stabilize wine and prevent protein haze formation after bottling. Currently many approaches are used to find an alternative because of bentonite's negative influence on color and aroma compounds. Nevertheless, the interaction of bentonite with wine proteins is not fully understood. Therefore, we analyzed the influence of a NaCa-combined bentonite on the protein content and composition of different wines showing a partial selectivity on protein adsorption. For example, glycosylated proteins were not removed by bentonite in notable amounts. Performing mass spectrometry we were able to demonstrate that 96% of class IV chitinase was adsorbed by bentonite, whereas varying adsorption behavior was observed for different thaumatin-like protein isoforms ranging from no removal to 98% of reduction. These differences may be explained by considering hydrophobicity surface of the proteins. Here, some differences are detected which might be an explanation for the better or worse adsorption. Our results demonstrate the importance to consider the isoforms of the potential haze-related proteins. Only the understanding of the interaction conditions of the wine component and their behavior with fining agents might result in the development of new strategies preventing protein haze formation.

Protein haze in white wines is a major technological and economic problem for the wine industry. In particular, an increased haze formation can be observed in wines from dry vintages. Therefore, field tests were carried out in steep slope vineyards planted with Riesling grapes over three dry growing seasons to study the effect of drought stress on the concentration of proteins in the resulting wines. Plots suffering from drought stress were compared with the surrounding drip-irrigated plots. Riesling grapes were processed into wines by conventional procedures. Protein amounts of the isolated wine colloids of the stressed samples (S) were always higher than those of the watered samples (W) (mean watered 13.8 ± 0.44, mean stressed 17.4 ± 0.40 g 100 g−1 isolated colloids). As a consequence, higher bentonite doses were needed to achieve protein haze stability of the drought-stressed treatments. Concerning the protein composition, there were no significant differences between stressed and watered treatments. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the molecular weights of proteins ranged from 12 to 75 kDa with an accumulation of chitinases and thaumatin-like proteins between 20 and 30 kDa. Concerning the protein composition, minor differences became obvious only between vintages but not between the stressed and watered samples. In-solution digest of proteins from Riesling grapes 2008 followed by LC-MSn and database research identified 15 proteins originating from grapes and 10 from the yeast Saccharomyces cerevisiae. In conclusion, our study demonstrates the importance to develop new strategies to prevent increased haze formation in wine under the predicted conditions of climate change. Prevention of drought stress by drip irrigation can be considered as one strategy to decrease protein amounts in wines.

Show your metal: l-Tyrosine is converted into the protective antioxidative polymer melanin in a sequence of reactions. In humans, the catalytic pathway starts with the tyrosinase HsTYR and two tyrosinase-related proteins HsTYRP1 and HsTYRP2. All three enzymes have the same active site but the latter two contain two zinc ions instead of copper ions.

Tyrosinasen ermoglichen die ortho-Hydroxylierung und Zwei-Elektronen-Oxidation von Monophenolen zu ortho-Chinonen. Catecholoxidasen katalysieren dagegen nur die Oxidation von Diphenolen. Obwohl diese Prozesse von signifikantem Interesse sind, ist die molekulare Basis des funktionellen Unterschiedes zwischen Tyrosinasen und Catecholoxidasen noch unklar. Vor kurzem wurde postuliert, dass eine Glutaminsaure und ein Asparagin ein konserviertes Wassermolekul binden und aktivieren, um dadurch Monophenole zu deprotonieren. Hier zeigen wir, dass eine Polyphenoloxidase, die nur eine Diphenolase-Aktivitat besitzt, durch die Einfuhrung eines Asparagins mittels Mutagenese in eine Tyrosinase umgewandelt werden kann. Das Asparagin und die konservierte Glutaminsaure sind fur eine korrekte Orientierung und Aktivierung des konservierten Wassermolekuls notwendig, damit es die Hydroxygruppe des Monophenols deprotonieren kann.

Tyrosinases mediate the ortho-hydroxylation and two-electron oxidation of monophenols to ortho-quinones. Catechol oxidases only catalyze the oxidation of diphenols. Although it is of significant interest, the origin of the functional discrimination between tyrosinases and catechol oxidases has been unclear. Recently, it has been postulated that a glutamate and an asparagine bind and activate a conserved water molecule towards deprotonation of monophenols. Here we demonstrate for the first time that a polyphenoloxidase, which exhibits only diphenolase activity, can be transformed to a tyrosinase by mutation to introduce an asparagine. The asparagine and a conserved glutamate are necessary to properly orient the conserved water in order to abstract a proton from the monophenol. These results provide direct evidence for the crucial importance of a proton shuttle for tyrosinase activity of type 3 copper proteins, allowing a consistent understanding of their different chemical reactivities.

Macromolecular oligomeric assemblies are involved in many biochemical processes of living organisms. The benefits of such assemblies in crowded cellular environments include increased reaction rates, efficient feedback regulation, cooperativity and protective functions. However, an atom‐level structural determination of large assemblies is challenging due to the size of the complex and the difference in binding affinities of the involved proteins. In this study, we propose a novel combinatorial greedy algorithm for assembling large oligomeric complexes from information on the approximate position of interaction interfaces of pairs of monomers in the complex. Prior information on complex symmetry is not required but rather the symmetry is inferred during assembly. We implement an efficient geometric score, the transformation match score, that bypasses the model ranking problems of state‐of‐the‐art scoring functions by scoring the similarity between the inferred dimers of the same monomer simultaneously with different binding partners in a (sub)complex with a set of pregenerated docking poses. We compiled a diverse benchmark set of 308 homo and heteromeric complexes containing 6 to 60 monomers. To explore the applicability of the method, we considered 48 sets of parameters and selected those three sets of parameters, for which the algorithm can correctly reconstruct the maximum number, namely 252 complexes (81.8%) in, at least one of the respective three runs. The crossvalidation coverage, that is, the mean fraction of correctly reconstructed benchmark complexes during crossvalidation, was 78.1%, which demonstrates the ability of the presented method to correctly reconstruct topology of a large variety of biological complexes. Proteins 2015; 83:1887–1899. © 2015 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.

Grape proteins are discussed to cause haze formation in bottled wine leading to economic problems. A common way to remove these proteins is bentonite fining. Although the efficiency of bentonite treatment is well known, the underlying molecular mechanism of the fining process in wine is not well understood. Two white wine varieties, Riesling and Pinot gris, were treated in the must stage with different types of bentonite to investigate their efficiencies. In all cases, bentonite treatment significantly reduced the overall protein concentration based on SDS-PAGE analysis, albeit individual proteins were differently affected. However, our data indicate that one protein band was completely removed independent of the bentonite type. Mass spectrometry analysis identified the major protein as glucan endo-1,3-β-glucosidase of Vitis vinifera, which was accompanied by low amounts of several other proteins. Using molecular modeling, we propose that this enzyme with its positively charged surface is adsorbed on the negatively charged lamella of bentonite during the fining process.

The characteristics of intestinal model fluids were investigated at conditions, which simulate the passage from the middle to the end of the duodenum. The formation and decay of liposomes and micelles in model bile fluids were studied, because of their role as an intermediate host for the resolution and uptake of hydrophobic drugs (BCS classes II, IV). The conditions, which may influence the formation of these nanoparticulate intermediates were studied, i.e., the lipid composition of the bile, the preparation method, the time of the passage through the modelled duodenum segment and the concentration, which results from the variable dilution of the bile by mixing with the transfer medium representing the fluid arriving from the stomach. The variation of the lecithin entity revealed an equivalence of Egg–lecithin of a high purity (99%) with its main component 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine POPC, while 1,2-di-oleoyl-sn-glycero-3-phosphocholine DOPC resulted in a slight delay of the micelle-liposome conversion. The FeSSIF preparation method was best with the sequential-film method, while the bile-film method yielded comparable results; the shake method showed a slightly different kinetics of the nanoparticle conversion. The time and concentration dependence of the formation and decay of lipidic nanoparticles indicates that these strongly depend on the passage time (speed) and bile dilution rate. The corresponding physiological conditions at healthy persons may vary in vivo individually and due to diseases. The studied conditions cover typical physiological conditions, which should be taken into consideration in the exploration of in vitro tests of formulations of hydrophobic drugs. Formation and decay of liposomes and micelles from bile in the duodenum was studied, due to their role as an intermediate for resolution and uptake of hydrophobic drugs (BCS II, IV). The physiological conditions studied may vary individually and due to diseases.

A novel homogenous core-shell formulation ‘AmbiShell’ of the hydrophobic antifungal polyene drug Amphotericin B was prepared from the pure drug, lecithin, and gelatin. The established ‘Hydrophilic Solubilisation Technology’ (HST) was used for embedding the lipophilic drug with lecithin and gelatin, yielding core-shell microparticles of 1 µm size. The particle size of the drug, the formulation, and the thickness of the coating were investigated by using dynamic light scattering (DLS), microscopy, UV–Visible spectroscopy and small angle neutron scattering (SANS). The drug in the formulation was not in the oligomer form but in a competent uptake form, as shown by the UV–Visible spectroscopy. The DLS and microscopy depicted the product to consist of spheres (∼1 µm). The SANS experiment using deuterium contrast matching of the drug cores revealed a thin layer span of 5.6 nm, which is similar, but slightly larger as compared to the biological lipid membranes. The finding offers opportunities for further improvements of the formulation, e.g., by surface modification. A soluble core-shell formulation ‘AmbiShell’ of the hydrophobic antifungal polyene drug Amphotericin B was generated from drug, lecithin, and gelatin. The span of the lipid–gelatin shell was estimated by neutron scattering SANS with deuterium contrast matching of the drug core.

Wine is produced from grapes which contain enzymes forming unfavorable dark polymers such as melanin, oxidizing proteins, and phenols which may influence their functions. Here we focus on the class of type-3 copper proteins presenting new insights on the structure, the occurrence during the ripening process and during wine making. Then, the reactivity of the enzymes on polyphenols from wine grapes are discussed and the possible consequences on health.

Phenolic components (PCs) are well-known for their positive impact on human health. In addition to their action as radical scavengers, they act as activators for the intrinsic cellular antioxidant system. Polyphenol oxidases (PPOs) such as tyrosinase and laccase catalyze the enzymatic oxidation of PCs and thus, can alter their scavenging and antioxidative capacity. In this study, oxidation by tryosinase was shown to increase the antioxidant capacity of many PCs, especially those that lack adjacent aromatic hydroxyl groups. In contrast, oxidation by laccase tended to decrease the antioxidant capacity of red wine and distinct PCs. This was clearly demonstrated for p-coumaric acid and resveratrol, which is associated with many health benefits. While oxidation by tyrosinase increased their antioxidant activity laccase treatment resulted in a decreased activity and also of that for red wines.

Hemocyanins are multimeric oxygen-transport proteins in the hemolymph of many arthropods and mollusks. The overall molecular architecture of arthropod and molluscan hemocyanin is very different, although they possess a similar binuclear type 3 copper center to bind oxygen in a side-on conformation. Gastropod hemocyanin is a 35 nm cylindrical didecamer (2 × 10-mer) based on a 400 kDa subunit. The latter is subdivided into eight paralogous “functional units” (FU-a to FU-h), each with an active site. FU-a to FU-f contribute to the cylinder wall, whereas FU-g and FU-h form the internal collar complex. Atomic structures of FU-e and FU-g, and a 9 A cryoEM structure of the 8 MDa didecamer are available. Recently, the structure of keyhole limpet hemocyanin FU-h (KLH1-h) was presented as a Cα-trace at 4 A resolution. Unlike the other seven FU types, FU-h contains an additional C-terminal domain with a cupredoxin-like fold. Because of the resolution limit of 4 A, in some loops, the course of the protein backbone could not be established with high certainty yet. Here, we present a refined atomic structure of FU-h (KLH1-h) obtained from low-resolution refinement, which unambiguously establishes the course of the polypeptide backbone and reveals the disulfide bridges as well as the orientation of bulky amino acids. © 2011 IUBMB IUBMB Life, 63(3): 183–187, 2011

Allergens from cockroaches cause major asthma-related health problems worldwide. Among them Per a 3 belongs to the most potent allergens. Although the sequences of some members of the Per a 3-family are known, their biochemical and biophysical properties have not been investigated. Here we present for the first time a thorough structural characterization of these allergens, which have recently been tested to induce an increase of allergy specific indicators in blood of Europeans. We isolated two Per a 3 isoforms, which occur freely dissolved in the hemolymph as hexamers with molecular masses of 465+/-25kDa (P II) and 512+/-25kDa (P I). Their sedimentation coefficients (S(20,W)) were determined to be 17.4+/-0.7 S (P II) and 19.0+/-0.9 S (P I), respectively. Sequence analysis revealed that P II consists of two subunit types known as allergens Per a 3.01 and Per a 3.0201, while PI consists of a new allergenic subunit type designated as Per a 3.03. A 3D model of the hexameric allergen Per a 3 was obtained by homology modelling. Almost all of the recently predicted 11 putative antigenic peptides and reported IgE-epitopes could be located on the surface of the hexamer, thus being freely accessible in the hexameric structure of the native molecules. We propose this might contribute to their allergic potential as well as their extreme stability with respect to temperature.

Active metal sites play a key role in the biochemistry of oxygen transport by hemocyanins. Observing the changes in the local electronic structure of the copper sites upon oxygenation is thus essen...

Summary Phenoloxidases (POs) occur in all organisms and are involved in skin and hair coloring in mammals, and initiating melanization in wound healing. Mutation or overexpression of PO can cause albinism or melanoma, respectively. SDS can convert inactive PO and the oxygen carrier hemocyanin (Hc) into enzymatically active PO. Here we present single-particle cryo-EM maps at subnanometer resolution and pseudoatomic models of the 24-oligomeric Hc from scorpion Pandinus imperator in resting and SDS-activated states. Our structural analyses led to a plausible mechanism of Hc enzyme PO activation: upon SDS activation, the intrinsically flexible Hc domain I twists away from domains II and III in each subunit, exposing the entrance to the active site; this movement is stabilized by enhanced interhexamer and interdodecamer interactions, particularly in the central linker subunits. This mechanism could be applicable to other type 3 copper proteins, as the active site is highly conserved.

α-Hemolysin from Staphylococcus aureus is secreted as a water-soluble monomer and assembles on membranes to oligomerize into a homo-heptameric, water-filled pore. These pores lead to lysis and cell death. Although the structure of the heptameric pore is solved by means of X-ray crystallography, structures of intermediate states—from the soluble monomer to all potential “pre-pore” structures—are yet unknown. Here, we propose a model of the monomeric α-hemolysin in solution based on molecular modeling, verified by small angle X-ray scattering data. This structure reveals details of the monomeric conformation of the α-hemolysin, for example inherent flexibility, along with definite differences in comparison to the structures used as templates. Proteins 2009. © 2008 Wiley-Liss, Inc.

We use photoemission electron microscopy in an X-ray transmission mode for full-field imaging of the X-ray absorption structure of copper in the respiratory metalloprotein hemocyanin KLH1. It contains 160 oxygen binding sites. Each site reversibly binds one molecule oxygen between two copper atoms. In our setup, hemocyanin is dissolved in aqueous solution and enclosed in an ultra-high vacuum compatible liquid sample cell with silicon nitride membranes. The local X-ray absorption structure of the liquid sample is converted into photoelectrons at the microscope side of the cell acting as a photocathode. In this way, different copper valencies are laterally distinguished under in vivo-like conditions, attributed to Cu(I) in the deoxy-state and Cu(II) in the oxy-state.

Phenoloxidases occur in almost all organisms, being essentially involved in various processes such as the immune response, wound healing, pigmentation and sclerotization in arthropods. Many hemocyanins are also capable of phenoloxidase activity after activation. Notably, in chelicerates, a phenoloxidase has not been identified in the hemolymph, and thus hemocyanin is assumed to be the physiological phenoloxidase in these animals. Although phenoloxidase activity has been shown for hemocyanin from several chelicerate species, a characterization of the enzymatic properties is still lacking. In this article, the enzymatic properties of activated hemocyanin from the tarantula Eurypelma californicum are reported, which was activated by SDS at concentrations above the critical micellar concentration. The activated state of Eurypelma hemocyanin is stable for several hours. Dopamine is a preferred substrate of activated hemocyanin. For dopamine, a K(M) value of 1.45 +/- 0.16 mm and strong substrate inhibition at high substrate concentrations were observed. Typical inhibitors of catecholoxidase, such as l-mimosine, kojic acid, tyramine, phenylthiourea and azide, also inhibit the phenoloxidase activity of activated hemocyanin. This indicates that the activated hemocyanin behaves as a normal phenoloxidase.

This review summarizes recent highlights of our joint work on the structure, evolution, and function of a family of highly complex proteins, the hemocyanins. They are blue-pigmented oxygen carriers, occurring freely dissolved in the hemolymph of many arthropods and molluscs. They are copper type-3 proteins and bind one dioxygen molecule between two copper atoms in a side-on coordination. They possess between 6 and 160 oxygen-binding sites, and some of them display the highest molecular cooperativity observed in nature. The functional properties of hemocyanins can be convincingly described by either the Monod-Wyman-Changeux (MWC) model or its hierarchical extension, the Nested MWC model; the latter takes into account the structural hierarchies in the oligomeric architecture. Recently, we applied these models to interpret the influence of allosteric effectors in detailed terms. Effectors shift the allosteric equilibria but have no influence on the oxygen affinities characterizing the various conformational states. We have shown that hemocyanins from species living at different environmental temperatures have a cooperativity optimum at the typical temperature of their natural habitat. Besides being oxygen carriers, some hemocyanins function as a phenoloxidase (tyrosinase/catecholoxidase) which, however, requires activation. Chelicerates such as spiders and scorpions lack a specific phenoloxidase, and in these animals activated hemocyanin might catalyse melanin synthesis in vivo. We propose a similar activation mechanism for arthropod hemocyanins, molluscan hemocyanins and tyrosinases: amino acid(s) that sterically block the access of phenolic compounds to the active site have to be removed. The catalysis mechanism itself can now be explained on the basis of the recently published crystal structure of a tyrosinase. In a series of recent publications, we presented the complete gene and primary structure of various hemocyanins from different molluscan classes. From these data, we deduced that the molluscan hemocyanin molecule evolved ca. 740 million years ago, prior to the separation of the extant molluscan classes. Our recent advances in the 3D cryo-electron microscopy of hemocyanins also allow considerable insight into the oligomeric architecture of these proteins of high molecular mass. In the case of molluscan hemocyanin, the structure of the wall and collar of the basic decamers is now rapidly becoming known in greater detail. In the case of arthropod hemocyanin, a 10-Å structure and molecular model of the Limulus 8 × 6mer shows the amino acids at the various interfaces between the eight hexamers, and reveals histidine-rich residue clusters that might be involved in transferring the conformational signals establishing cooperative oxygen binding.

Tyrosinases are the first and rate-limiting enzymes in the synthesis of melanin pigments responsible for colouring hair, skin and eyes. Mutation of tyrosinases often decreases melanin production resulting in albinism, but the effects are not always understood at the molecular level. Homology modelling of mouse tyrosinase based on recently published crystal structures of non-mammalian tyrosinases provides an active site model accounting for loss-of-function mutations. According to the model, the copper-binding histidines are located in a helix bundle comprising four densely packed helices. A loop containing residues M374, S375 and V377 connects the CuA and CuB centres, with the peptide oxygens of M374 and V377 serving as hydrogen acceptors for the NH-groups of the imidazole rings of the copper-binding His367 and His180. Therefore, this loop is essential for the stability of the active site architecture. A double substitution (374)MS(375) --> (374)GG(375) or a single M374G mutation lead to a local perturbation of the protein matrix at the active site affecting the orientation of the H367 side chain, that may be unable to bind CuB reliably, resulting in loss of activity. The model also accounts for loss of function in two naturally occurring albino mutations, S380P and V393F. The hydroxyl group in S380 contributes to the correct orientation of M374, and the substitution of V393 for a bulkier phenylalanine sterically impedes correct side chain packing at the active site. Therefore, our model explains the mechanistic necessity for conservation of not only active site histidines but also adjacent amino acids in tyrosinase.

This review presents the common features and differences of the type 3 copper proteins with respect to their structure and function. In spite of these differences a common mechanism of activation and catalysis seems to have been preserved throughout evolution. In all cases the inactive proenzymes such as tyrosinase and catecholoxidase are activated by removal of an amino acid blocking the entrance channel to the active site. No other modification at the active site seems to be necessary to enable catalytic activity. Hemocyanins, the oxygen carriers in many invertebrates, also behave as silent inactive enzymes and can be activated in the same way. The molecular basis of the catalytic process is presented based on recent crystal structures of tyrosinase and hemocyanin. Minor conformational differences at the active site seem to decide about whether the active site is only able to oxidize diphenols as in catecholoxidase or if it is also able to o-hydroxylate monophenols as in tyrosinase.

The structural properties of the hemocyanin isolated from the Mediterranean mud shrimp, Upogebia pusilla (Decapoda: Thalassinidea), were investigated. Our intent was to make use of the U. pusilla case to perform a structural comparison between crustacean and chelicerate 4×6-meric hemocyanins. The thalassinidean hemocyanin appears similar in size but different in structural organization compared to the chelicerate 4×6-mer. Ultracentrifuge analyses on the purified protein revealed a sedimentation coefficient of 39S, typical of 4×6 hemocyanins. Electron micrographs are in agreement with a model in which four 2×6-meric building blocks are arranged in a tetrahedron-like quaternary structure and not in the quasi-square-planar orientation characteristic of the chelicerate protein. Size-exclusion chromatography-fast protein chromatography analysis showed elevated instability of the protein in absence of divalent ions or at pH values higher than 8.0. This analysis also shows that the dissociation of the U. pusilla 4×6-meric hemocyanin into hexamers occurs without any intermediate 2×6-meric state, in contrast with the dissociation profile of the chelicerate protein exhibiting several dissociation intermediates. The oxygen-binding properties of U. pusilla hemocyanin were studied to disclose possible effects by the typical allosteric effectors that modulate the functional properties of crustacean hemocyanin. A marked Bohr and lactate effect, but no significant influence of urate, on the oxygen affinity of U. pusilla hemocyanin were found. © 2007 Elsevier B.V. All rights reserved.

Polysaccharides are the major high-molecular weight components of wines. In contrast, proteins occur only in small amounts in wine, but contribute to haze formation. The detailed mechanism of aggregation of these proteins, especially in combination with other wine components, remains unclear. This study demonstrates the different aggregation behavior between a buffer and a model wine system by dynamic light scattering. Arabinogalactan-protein, for example, shows an increased aggregation in the model wine system, while in the buffer system a reducing effect is observed. Thus, we could show the importance to examine the behavior of wine additives under conditions close to reality, instead of simpler buffer systems. Additional experiments on melting points of wine proteins reveal that only some isoforms of thaumatin-like proteins and chitinases are involved in haze formation. We can confirm interactions between polysaccharides and proteins, but none of these polysaccharides is able to prevent haze in wine.

Background In a recently performed survey with 4000 randomly selected persons, 68 (7.2 %) of 948 respondents reported intolerance and/or allergy-like symptoms to wine. The aim of this study was to analyze whether a real sensitization to wine proteins could be confirmed by diagnostic and/or immunological settings. Findings For this purpose, 19 subjects with self-reported intolerance to wine of the invited subjects and 10 controls without a history of intolerance participated in an allergological examination (skin prick test, ImmunoCAP for determination of specific IgE antibodies, CAST for testing basophil activation, ImmunoBlot for testing specificity of IgE-antibodies). For the allergological work-up red and white grapes, selected wines, and the purified lipid transfer protein (LTP), a known grape allergen, were used. 7 subjects showed evidence of IgE sensitization to wine or grape extracts, including one control. One participant with symptoms of intolerance showed a positive skin prick test to red grape, a positive ImmunoCAP to grape, a positive cellular antigen stimulation test (CAST) and inhibition of Western blot by removal of cross-reactive carbohydrate determinants (CCD). Conclusion The presented study focused on the grape protein-related IgE-mediated cause of intolerance to wine (true allergy) and not on other wine components or fining agents (other forms of intolerance). A sensitization to grape and wine proteins was observed in our cohort. In one case, this reactivity could be explained by cross-reactivity to CCD. The results of this pilot study need to be validated in greater cohorts.

The high molecular mass respiratory protein of the tarantula Eurypelma californicum, a 4 x 6-mer hemocyanin, was investigated by atomic force microscopy (AFM). Various substrates and methods were evaluated for immobilization of individual hemocyanin molecules on a solid surface. Samples were imaged after physisorption on mica and self-assembled monolayers, and after chemisorption on Au(111) and N-hydroxy-succinimide (NHS) functionalized surfaces. AFM measurements were carried out preferable in solution and contact mode, but also in Tapping mode and on air-dried samples. Adsorption of the protein on mica followed by drying and carrying out the measurements in Tapping mode gave the best results. In the AFM images the four hexamers of the native 4 x 6-mer hemocyanin have been defined. The results were compared with independent available structural data and represent a validation case for this technique applied for the first time on such giant and complex molecules. As observable in images taken by transmission electron microscopy and also proposed from SAXS data, 4 x 6-mers could be found where the half-molecules are tilted against each other. This study is a step in resolving conformational heterogeneities, involved in oxygen binding of hemocyanins, at the single-molecule level by AFM.

The molecular masses of macromolecules and subunits of the extracellular hemoglobin from the fresh-water crustacean Daphnia magna were determined by analytical ultracentrifugation, multiangle laser light scattering and electrospray ionization mass spectrometry. The hemoglobins from hypoxia-incubated, hemoglobin-rich and normoxia-incubated, hemoglobin-poor Daphnia magna were analyzed separately. The sedimentation coefficient of the macromolecule was 17.4 +/- 0.1 S, and its molecular mass was 583 kDa (hemoglobin-rich animals) determined by AUC and 590.4 +/- 11.1 kDa (hemoglobin-rich animals) and 597.5 +/- 49 kDa (hemoglobin-poor animals), respectively, determined by multiangle laser light scattering. Measurements of the hemoglobin subunit mass of hemoglobin-rich animals by electrospray ionization mass spectrometry revealed a significant peak at 36.482 +/- 0.0015 kDa, i.e. 37.715 kDa including two heme groups. The hemoglobin subunits are modified by O-linked glycosylation in the pre-A segments of domains 1. No evidence for phosphorylation of hemoglobin subunits was found. The subunit migration behavior during SDS/PAGE was shown to be influenced by the buffer system used (Tris versus phosphate). The subunit mass heterogeneity found using Tris buffering can be explained by glycosylation of hemoglobin subunits. Based on molecular mass information, Daphnia magna hemoglobin is demonstrated to consist of 16 subunits. The quaternary structure of the Daphnia magna hemoglobin macromolecule was assessed by three-dimensional reconstructions via single-particle analysis based on negatively stained electron microscopic specimens. It turned out to be much more complex than hitherto proposed: it displays D4 symmetry with a diameter of approximately 12 nm and a height of about 8 nm.

In many crustaceans, changing concentrations of several low molecular weight compounds modulates hemocyanin oxygen binding, resulting in lower or higher oxygen affinities of the pigment. The nonphysiological effector caffeine and the physiological modulator urate, the latter accumulating in the hemolymph of the lobster Homarus vulgaris during hypoxia, increase hemocyanin oxygen affinity and decrease cooperativity of oxygen binding. To derive a model that describes the mechanism of allosteric interaction between hemocyanin and oxygen in the presence of urate or caffeine, studies of oxygen, urate, and caffeine binding to hemocyanin were performed. Exposure of lobster hemocyanin to various pH values between 7.25 and 8.15 resulted in a decrease of p50. In this pH interval, p50 decreases from 95 to 11 Torr without effectors and from 49 to 6 Torr and from 34 to 5 Torr in the presence of 1 mM urate or caffeine, respectively. Thus, the allosteric effects induced by protons and urate or caffeine are coupled. In contrast, isothermal titration calorimetry did not reveal any differences in binding enthalpy (DeltaH degrees ) for urate or caffeine under either normoxic or hypoxic conditions at different pH values. Despite these apparently conflicting results, they can be explained by the nested MWC model if two different types of modulator binding sites are assumed, an allosteric and a nonallosteric type of site. Simulations of in vivo conditions with this model indicate that the naturally occurring modulator urate is physiologically relevant in H. vulgaris only during hypoxic conditions, i.e., either during environmental oxygen limitation or extensive exercise.

The conformational distribution of the N-terminal domain of the major light-harvesting chlorophyll a/b protein (LHCIIb) has been characterized by electron-electron double resonance yielding distances between spin labels placed in various domains of the protein. Distance distributions involving residue 3 near the N terminus turned out to be bimodal, revealing that this domain, which is involved in regulatory functions such as balancing the energy flow through photosystems (PS) I and II, exists in at least two conformational states. Models of the conformational sub-ensembles were generated on the basis of experimental distance restraints from measurements on LHCIIb monomers and then checked for consistency with the experimental distance distribution between residues 3 in trimers. Only models where residue 3 is located above the core of the protein and extends into the aqueous phase on the stromal side fit the trimer data. In the other state, which consequently is populated only in monomers, the N-terminal domain extends sideways from the protein core. The two conformational states may correspond to two functional states of LHCIIb, namely trimeric LHCIIb associated with PSII in stacked thylakoid membranes and presumably monomeric LHCIIb associated with PSI in nonstacked thylakoids. The switch between these two is known to be triggered by phosphorylation of Thr-6. A similar phosphorylation-induced conformational change of the N-terminal domain has been observed by others in bovine annexin IV which, due to the conformational switch, also loses its membrane-aggregating property.

Hemocyanins are large respiratory proteins of arthropods and mollusks, which bind oxygen with very high cooperativity. Here, we investigated the relationship between oxygen binding and structural changes of the 24-mer tarantula hemocyanin. Oxygen binding of the hemocyanin was detected following the fluorescence intensity of the intrinsic tryptophans. Under the same conditions, structural changes were monitored by the non-covalently bound fluorescence probe Prodan (6-propionyl-2-(dimethylamino)-naphthalene), which is very sensitive to its surroundings. Upon oxygen binding of the hemocyanin a red shift of 5 nm in the emission maximum of the label was observed. A comparison of oxygen binding curves recorded with tryptophan and Prodan emission revealed that structural changes in tarantula hemocyanin lag behind oxygen binding at the beginning of oxygenation. Analyses based on the nested two-state model, which describes cooperative oxygen binding of hemocyanins, indicated that the transition monitored by Prodan emission is closely related to one of the four conformations (rR) predicted for the allosteric unit. Earlier, the allosteric unit of tarantula hemocyanin was found to be the 12-mer half-molecule. Here, fluorescence titration revealed that the number of Prodan binding sites/24-mer tarantula hemocyanin is approximately 2, matching the number of allosteric units/hemocyanin. Based on the agreement between oxygen binding curves and fluorescence titration we concluded that Prodan monitors a conformational transition of the allosteric unit.

The hemocyanin of the tiger shrimp, Penaeus monodon, was investigated with respect to stability and oxygen binding. While hexamers occur as a major component, dodecamers and traces of higher aggregates are also found. Both the hexamers and dodecamers were found to be extremely stable against dissociation at high pH, independently of the presence of calcium ions, in contrast to the known crustacean hemocyanins. This could be caused by only a few additional noncovalent interactions between amino acids located at the subunit-subunit interfaces. Based on X-ray structures and sequence alignments of related hemocyanins, the particular amino acids are identified. At all pH values, the p50 and Bohr coefficients of the hexamers are twice as high as those of dodecamers. While the oxygen binding of hexamers from crustaceans can normally be described by a simple two-state model, an additional conformational state is needed to describe the oxygen-binding behaviour of Penaeus monodon hemocyanin within the pH range of 7.0 to 8.5. The dodecamers bind oxygen according to the nested Monod-Whyman-Changeaux (MWC) model, as observed for the same aggregation states of other hemocyanins. The oxygen-binding properties of both the hexameric and dodecameric hemocyanins guarantee an efficient supply of the animal with oxygen, with respect to the ratio between their concentrations. It seems that under normoxic conditions, hexamers play the major role. Under hypoxic conditions, the hexamers are expected not to be completely loaded with oxygen. Here, the dodecamers are supposed to be responsible for the oxygen supply.

For decades the respiratory protein keyhole limpet hemocyanin (KLH1) from the marine gastropod Megathura crenulata has been used widely as a potent immunostimulant, useful hapten carrier, and valuable agent in the treatment of bladder carcinoma. Although much information on the immunological properties of KLH1 is available, biochemical and structural data are still incomplete. Small-angle x-ray scattering revealed the existence of two conformations, an oxy state being slightly more compact than the deoxy state. Based on small-angle scattering curves, a newly developed Monte Carlo algorithm delivered a surface representation of proteins. The massive changes of the surfaces of reconstructed didecameric KLH1 molecules are explained as a twist of the two non-covalently associated decameric half-molecules. Upon oxygenation, the KLH1 molecule becomes longer and skinnier. This study provides the first real evidence how a molluscan hemocyanin changes conformation during an allosteric transition.

Hemocyanin from the tarantula Eurypelma californicum is a large respiratory protein with an exceptional high cooperativity. In contrast to hemocyanins from other species, no physiological allosteric effectors other than protons have been identified so far for this 24-meric oligomer. Here we report for the first time the mediating effects of water activity on the oxygen binding properties of a hemocyanin. Oxygen binding curves were measured in presence of several concentrations of glycine and sucrose since both substances reduce water activity. A pronounced shift of the p(50) was observed in both cases but in different directions: adding sucrose shifts the p(50) towards lower values whereas presence of glycine shows the same tendency as for human hemoglobin. Furthermore, prolonged incubation in sucrose slightly distorts the oxygen binding characteristics of spider hemocyanin. Therefore, only the influence of glycine was further analysed. An analysis based on the nested MWC model indicates, that presence of glycine leads to a preferential population of the two states with lower oxygen affinity (tR and tT) compared to the high affinity states rT and rR. The results corroborate the presence of hierarchically organized interactions in this hemocyanin.