All hierarchical levels are involved in conformational transitions of the 4 x 6-meric tarantula hemocyanin upon oxygenation

The respiratory protein of the tarantula Eurypelma californicum is a 4 x 6-meric hemocyanin that binds oxygen with high cooperativity. This requires the existence of different conformations which have been confirmed by small angle X-ray scattering (SAXS). Here we present reconstructed 3D-models of the oxy- and deoxy-forms of tarantula hemocyanins, as obtained by fitting small angle X-rays scattering curves on the basis of known X-ray structures and electron microscopy of related hemocyanins. For the first time, the involvement of movements at all levels of the quaternary structure was confirmed for an arthropod hemocyanin upon oxygenation. The two identical 2 x 6-meric half-molecules of the native 4 x 6-mer were shifted in the oxy-state along each other compared with the deoxy-state by about 14 A. In addition, the angle between the two 2 x 6-meric half-molecules increased by 13 degrees. Within these 2 x 6-mers the two hexamers were rotated against each other by about 26 degrees with respect to the deoxy-state. In addition, the distance between the two trimers of each hexamer increased upon oxygenation by about 2.5 A. These strongly coupled movements are based on the particular hierarchical structure of the 4 x 6-mer. It also shows a concept of allosteric interaction in hierarchically assembled proteins to guarantee the involvement of all subunits of a native oligomer to establish very high Hill coefficients.     

Three-dimensional reconstruction of native Androctonus australis hemocyanin

A sample of native 4 x 6-meric hemocyanin of Androctonus australis was negatively stained with the double-layer technique, and was observed by transmission electron microscopy under low-dose conditions with a 50 degree and 0 degree tilt. The three-dimensional reconstruction method from "Single-exposure, random conical tilt series" was then applied. Independent three-dimensional reconstructions were obtained from the top, side and 45 degree views. Despite a pronounced flattening effect, presumably due to the specimen preparation technique, the positions of the 24 subunits composing the oligomer were unequivocally determined. This experiment definitely solves the problem of the architectural organization of the subunits in the cheliceratan 4 x 6-meric hemocyanins. Moreover, distinction between the flip and flop faces and an attenuated rocking effect were observed.     

The molecular heterogeneity of hemocyanin: Structural and functional properties of the 4x6-meric protein of Upogebia pusilla (Crustacea)

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 4x6-meric hemocyanins. The thalassinidean hemocyanin appears similar in size but different in structural organization compared to the chelicerate 4x6-mer. Ultracentrifuge analyses on the purified protein revealed a sedimentation coefficient of 39S, typical of 4x6 hemocyanins. Electron micrographs are in agreement with a model in which four 2x6-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 4x6-meric hemocyanin into hexamers occurs without any intermediate 2x6-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.     

Differential regulation of hexameric and dodecameric hemocyanin from A. leptodactylus

The oxygen binding properties of hemocyanins are regulated on a short time scale by effectors such as l-lactate, urate and protons, and on longer time scales by expression of the different types of subunits. For Astacus leptodactylus it was shown previously that acclimation to higher temperatures leads to increased levels of a 6-meric hemocyanin species, whereas at lower temperatures the 12-meric form prevails. Here we show that the temperature dependence of the two forms supports the idea, that the maintenance of high affinity towards oxygen is the driving force for the differential expression of these hemocyanins. Furthermore, the two different types of hemocyanin differ not only in the affinity to oxygen, but also with respect to their interaction with l-lactate: while the 12-meric form displays a normal shift in oxygen affinity upon the addition of l-lactate this allosteric regulation is absent in the 6-meric form. Exclusive binding of l-lactate to the 12-meric form was supported by isothermal titration calorimetry. These results indicate that l-lactate binds either at the interface between the two hexamers or at subunit α′ which is responsible for the formation of the 12-mers and is not present in the 6-meric form. Urate has a comparable effect on the oxygen affinity of 6-meric and 12-meric forms and also binds to a similar extent to the oxygenated state as determined by isothermal titration calorimetry. Thus, urate and l-lactate do not seem to share the same binding sites. Interestingly, urate binding sites with no allosteric effect seem to exist, which is unusual. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.     

Unusual oxygen binding behavior of a 24-meric crustacean hemocyanin

Hemocyanins from Crustacea usually are found as 1 × 6 or 2 × 6-meric assemblies. An exception is the hemocyanin isolated from thalassinidean shrimps where the main component is a 24-meric structure. Our analysis of oxygen binding data of the thalassinidean shrimp Upogebia pusilla based on a three-state MWC-model revealed that despite the 24-meric structure the functional properties can be described very well based on the hexamer as allosteric unit. In contrast to the hemocyanins from other thalassinidean shrimps the oxygen affinity of hemocyanin from U. pusilla is increased upon addition of l-lactate. A particular feature of this hemocyanin seems to be that l-lactate already enhances oxygen affinity under resting conditions which possibly compensates the rather low intrinsic affinity observed in absence of l-lactate. The fast rate of oxygen dissociation might indicate that in this hemocyanin a higher cooperativity is less important than a fast response of saturation level to changes in oxygen concentration.     

A potential role for water in the modulation of oxygen-binding by tarantula hemocyanin

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.     

Two types of urate binding sites on hemocyanin from the crayfish Astacus leptodactylus: an ITC study

The oxygen binding behaviour of hemocyanins from Crustacea is regulated by small organic compounds such as urate and -lactate. We investigated the binding characteristics of urate and the related compound caffeine to the 2×6-meric hemocyanin of A. leptodactylus under fully oxygenated conditions employing isothermal titration calorimetry (ITC). An analysis of urate and caffeine binding based on a model of n identical binding sites resulted in approximately four binding sites for caffeine and eight for urate. This result suggests that the binding process for these effectors is more complex than this most simple model. Therefore, we introduced a number of alternative models. Displacement experiments helped to select the appropriate model. Based on these experiments, at least two different types of binding sites for urate and caffeine exist on the 2×6-meric hemocyanin of A. leptodactylus. The two binding sites differ strongly in their specificity towards the two analogues. It can be hypothesized that two different subunit types (β and γ) are responsible for the two types of binding sites.     

The allosteric effector l-lactate induces a conformational change of 2x6-meric lobster hemocyanin in the oxy state as revealed by small angle x-ray scattering

Hemocyanins are multisubunit respiratory proteins found in many invertebrates. They bind oxygen highly cooperatively. However, not much is known about the structural basis of this behavior. We studied the influence of the physiological allosteric effector l-lactate on the oxygenated quaternary structure of the 2x6-meric hemocyanin from the lobster Homarus americanus employing small angle x-ray scattering (SAXS). The presence of 20 mm l-lactate resulted in different scattering curves compared with those obtained in the absence of l-lactate. The distance distribution functions p(r) indicated a more compact molecule in presence of l-lactate, which is also reflected in a reduction of the radius of gyration by about 0.2 nm (3%). Thus, we show for the first time on a structural basis that a hemocyanin in the oxy state can adopt two different conformations. This is as predicted from the analysis of oxygen binding curves according to the "nesting" model. A comparison of the distance distribution functions p(r) obtained from SAXS with those deduced from electron microscopy revealed large differences. The distance between the two hexamers as deduced from electron microscopy has to be shortened by up to 1.1 nm to agree well with the small angle x-ray curves.     

Allosteric oxygen-binding properties of reassembled tarantula (Eurypelma californicum) hemocyanin with incorporated apo- or met-subunits

4x6-meric hemocyanin of the tarantula Eurypelma californicum was dissociated into subunits; one type of subunit was removed by immunoaffinity chromatography and replaced by its apo- or met-form. The mixture was reassembled and the reconstituted 4x6-mers were isolated. This was performed for subunits a, bc, d, e, f and g, respectively. It was verified by crossed immunoelectrophoresis that each type of subunit including the modified one, was incorporated in the reassembled 4x6-mers. Oxygen binding curves of the purified reconstituted 4x6-mers were recorded at different pH values (pH 7.0-9.0; 20 degrees C). Half-saturation pressures (P50) and the cooperativities were calculated using unmodified, reassembled 4x6-mers as reference. In all cases, incorporation of a met-subunit increased oxygen affinity. In contrast, incorporation of an apo-subunit either slightly decreased oxygen affinity (bc, f and d) or had no detected influence (others). The Bohr effect remained more or less unchanged in every case. Cooperativity was generally decreased. The met-modification of subunit d had the strongest effect. No significant differences could be observed between the respective met- and apomodification, except for experiments with subunit d. Generally, the value of hmax exceeded h50 by a factor of 1.3 to 1.5. pH-sensitivity of cooperativity was distinctly influenced depending on the modified subunit. The strongest effect was observed for subunit bc. Our results demonstrate, for the first time, that each subunit of tarantula hemocyanin is involved in the allosteric processes. Apparently, they uniformly contribute to oxygen affinity and Bohr effect, but distinctly to cooperativity and pH sensitivity of the latter.     

Crystallization and preliminary analysis of crystals of the 24-meric hemocyanin of the emperor scorpion (Pandinus imperator)

Hemocyanins are giant oxygen transport proteins found in the hemolymph of several invertebrate phyla. They constitute giant multimeric molecules whose size range up to that of cell organelles such as ribosomes or even small viruses. Oxygen is reversibly bound by hemocyanins at binuclear copper centers. Subunit interactions within the multisubunit hemocyanin complex lead to diverse allosteric effects such as the highest cooperativity for oxygen binding found in nature. Crystal structures of a native hemocyanin oligomer larger than a hexameric substructure have not been published until now. We report for the first time growth and preliminary analysis of crystals of the 24-meric hemocyanin (M_W = 1.8 MDa) of emperor scorpion (Pandinus imperator), which diffract to a resolution of 6.5 Å. The crystals are monoclinc with space group C 1 2 1 and cell dimensions a = 311.61 Å, b = 246.58 Å and c = 251.10 Å (α = 90.00°, β = 90.02°, γ = 90.00°). The asymmetric unit contains one molecule of the 24-meric hemocyanin and the solvent content of the crystals is 56%. A preliminary analysis of the hemocyanin structure reveals that emperor scorpion hemocyanin crystallizes in the same oxygenated conformation, which is also present in solution as previously shown by cryo-EM reconstruction and small angle x-ray scattering experiments.     

Stability of a 24-meric homopolymer: comparative studies of assembly-defective mutants of Rhodobacter capsulatus bacterioferritin and the native protein

The stability of Rhodobacter capsulatus bacterioferritin, a 24-meric homopolymer, toward denaturation on variation in pH and temperature, and increasing concentrations of urea and guanidine.HCl was investigated with native PAGE, and CD and fluorescence spectroscopies. With temperature and urea, the wild-type protein denatured without discernible intermediates in the equilibrium experiments, but with guanidine.HCl (Gnd.HCl) one or more intermediate species were apparent at relatively low Gnd.HCl concentrations. Dissociated subunit monomers, or aggregates smaller than 24-mers containing the high alpha-helical content characteristic of the native protein were not obtained at any pH without a high proportion of the 24-mer being present, and taken together with the other denaturation experiments and the construction of stable subunit dimers by site-directed mutagenesis, this observation indicates that folding of the bacterioferritin monomer could be coupled to its association into a dimer. Glu 128 and Glu 135 were replaced by alanine and arginine in a series of mutants to determine their role in stabilizing the 24-meric oligomer. The Glu128Ala, Glu135Ala and Glu135Arg variants retained a 24-meric structure, but the Glu128Ala/Glu135Ala and Glu128Arg/Glu135Arg variants were stable subunit dimers. CD spectra of the Glu135Arg, Glu128Ala/Glu135Ala, and Glu128Arg/Glu135Arg variants showed that they retained the high alpha-helical content of the wild-type protein. The 24-meric Glu135Arg variant was less stable than the wild-type protein (T(m), [Urea](50%) and [Gnd.HCl](50%) of 59 degrees C, 4.9 M and 3.2 M compared with 73 degrees C, approximately 8 M and 4.3 M, respectively), and the dimeric Glu128Arg/Glu135Arg variant was less stable still (T(m), [Urea](50%) and [Gnd.HCl](50%) of 43 degrees C, approximately 3.2 M and 1.8 M, respectively). The differences in stability are roughly additive, indicating that the salt-bridges formed by Glu 128 and Glu 135 in the native oligomer, with Arg 61 and the amino-terminal amine of neighboring subunits, respectively, contribute equally to the stability of the subunit assembly. The additivity and assembly states of the variant proteins suggest that the interactions involving Glu 128 and Glu 135 contribute significantly to stabilizing the 24-mer relative to the subunit dimer.     

Real-time oligonucleotide hybridization kinetics monitored by resonant mirror technique

The kinetics of hybridization of 11-meric and 14-meric oligonucleotides, dTGGGAAGAGGG (ODN-11) and dTGGGAAGAGG GTCA (ODN-14), with 14-meric oligonucleotide dpTGACCCTCT TCCCA (p14) attached to the surface of a cuvette was studied by the resonant mirror method. The treatment of the experimental curves with exponential equations leads to the following values for association (kas) and dissociation (kdis) rate constants at 25 degrees C: kas = 219 +/- 39 and 183 +/- 162 M-1 s-1, kdis = (2.0 +/- 0.4) x 10(-3) and (4 +/- 1) x 10(-4) s-1 for the duplexes (p14) x (ODN-11) and p14 x (ODN-14), respectively. The oligonucleotide dTGCCTTGAATGGGAA GAGGGTCA (ODN-23), which forms a hairpin structure, does not associate with p14. The data were compared with the results of melting curve detection and temperature-jump experiments. The association rate constants for ODN-11 and ODN-14 are much slower than those values in homogeneous aqueous solution. The dissociation rate constants have the same magnitude values as estimated by using association constants measured from melting curves but differ from the values estimated in temperature-jump experiments.     

The oxygen-binding modulation of hemocyanin from the Southern spiny lobster <Emphasis Type="Italic">Palinurus gilchristi</Emphasis>

Arthropod hemocyanins transport and store oxygen and are composed of six subunits, or multiples thereof depending on the species. Palinurus gilchristi hemocyanin is found only as 1 × 6-mers, as normally occurs in spiny lobsters. An alkaline pH and removal of calcium ions induce a wholly reversible dissociation into monomers. The oxygen-binding properties of 1 × 6-meric hemocyanin from P. gilchristi were investigated with respect to pH and modulating effect exerted by calcium, lactate and urate. The oxygen affinity was highly affected by pH in the presence of calcium ions, while in its absence the Bohr coefficient became 60% lower. The protein is insensitive to lactate, but affected by urate which markedly increased hemocyanin–oxygen affinity, acting as the physiological major positive effector. Calcium ions decrease oxygen affinity at low concentration range (0–1 mM), while as concentration becomes higher than 100 mM, the oxygen affinity increases, indicating the presence of two independent types of calcium-binding sites with high and low affinity, respectively. The previous hypothesis, that the presence of high-affinity binding sites in addition to low affinity ones could be a characteristic feature of Palinuran hemocyanins, has been tested by analyzing, with respect to calcium–hemocyanin interaction, three other species belonging to Palinura.     

Review: Ontogeny of the oxygen transport in the blood]

The biological importance of the oxygen affinity of the tetrameric haemoglobin molecule and its dependency from co-factors is described. Oxygen affinity variations during development from embryonic to adult values are discussed in respect to oxygen uptake from maternal blood via placenta, in lungs and gills as well as to oxygen delivery into tissues. Comparative aspects of the molecular mechanisms leading to affinity changes are presented.     

Comparison of 4 × 6-meric hemocyanins from three different arthropods using computer alignment and correspondence analysis

We have compared 4 × 6-meric hemocyanin molecules from Androctonus australis and Eurypelma californicum with the similar 4 × 6-meric half-molecules of Limulus polyphemus hemocyanin. This comparison was performed using multivariate statistical analysis applied to computer-aligned electron microscopical images of the molecules: a method that was recently proposed by van Heel & Frank (1980,1981).Our study shows that the molecules are very similar indeed, and that the evidence for a tetrameric arrangement of the hexamers found in 4 × 6-meric Limulus molecules (van Heel & Frank, 1980,1981) also holds for Androctonus and Eurypelma hemocyanin. The model proposed for this molecule by Lamy et al. (1981) is therefore based on correct assumptions.In addition, our study shows that correspondence analysis, as a method of multivariate statistical analysis, is capable of detecting subtle differences between similar structures as well as differences in preparative conditions, effects that were hitherto not accessible to quantitative analysis.     

Hemocyanin from E. californicum encapsulated in silica gels: oxygen binding and conformational states

Cooperativity depends on the existence of equilibria among functionally distinct conformational states that are affected by homo and heterotropic effectors. In order to isolate the quaternary conformations of hemocyanin from E. californicum, the 24-meric giant protein was encapsulated in wet, nanoporous silica gels, either in the absence or presence of oxygen. The deoxy- and oxy-hemocyanin gels exhibit a p50 for oxygen of 11 and 2.5 torr, respectively, values in close agreement with those for hemocyanin in solution. The observed Hill coefficients are lower than unity, indicating a conformational heterogeneity within each locked conformational state, a finding in agreement with the assumption that at least four conformational states are required to explain the oxygen binding properties of E. californicum hemocyanin in solution.     

Structural basis of the iron storage function of frataxin from single-particle reconstruction of the iron-loaded oligomer

The mitochondrial protein frataxin plays a central role in mitochondrial iron homeostasis, and frataxin deficiency is responsible for Friedreich ataxia, a neurodegenerative and cardiac disease that affects 1 in 40000 children. Here we present a single-particle reconstruction from cryoelectron microscopic images of iron-loaded 24-subunit oligomeric frataxin particles at 13 and 17 A resolution. Computer-aided classification of particle images showed heterogeneity in particle size, which was hypothesized to result from gradual accumulation of iron within the core structure. Thus, two reconstructions were created from two classes of particles with iron cores of different sizes. The reconstructions show the iron core of frataxin for the first time. Compared to the previous reconstruction of iron-free particles from negatively stained images, the higher resolution of the present reconstruction allowed a more reliable analysis of the overall three-dimensional structure of the 24-meric assembly. This was done after docking the X-ray structure of the frataxin trimer into the EM reconstruction. The structure revealed a close proximity of the suggested ferroxidation sites of different monomers to the site proposed to serve in iron nucleation and mineralization. The model also assigns a new role to the N-terminal helix of frataxin in controlling the channel at the 4-fold axis of the 24-subunit oligomer. The reconstructions show that, together with some common features, frataxin has several unique features which distinguish it from ferritin. These include the overall organization of the oligomers, the way they are stabilized, and the mechanisms of iron core nucleation.     

Synthesis and characterization of soluble concanavalin A oligomer

Concanavalin A, (Con A, MW 26,500/monomer unit) was crosslinked with glutaraldehyde to form soluble, high-molecular-weight (larger than MW 300,000) Con A Oligomers. After filtration to remove insoluble and low-molecular-weight portions (below 300,000 daltons), the size and molecular-weight distribution were characterized by laser light scattering and gel-filtration chromatography. The molecular-size determined by laser light scattering ranged from 870 to 4070 A, while the molecular weight determined by gel chromatography ranged from 6 x 10(5) to higher than 2 x 10(6) daltons. The affinity and kinetics of Con A oligomer binding to polysaccharide (glycogen) were evaluated by precipitation test and turbidity development, respectively. The binding with glycogen was strongest at neutral pH and showed similar activity to unmodified Con A molecules. The binding constants of alpha-D-glucose and succinyl-aminophenyl alpha-D glucopyranoside-insulin to Con A oligomer were 1.0 x 10(3)M(-1) and 4.5 x 10(4)M(-1), respectively and the binding capacity of the oligomer was nearly 85% to 95% of monomeric Con A. The complexes of saccharides and soluble Con A oligomer were stable for at least 7 days. (c) 1993 Wiley & Sons, Inc.