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The quaternary structure of the (4 X 6)-mer hemocyanin from the Scorpion Androctonus australis previously published [Lamy, J., Bijlholt, M. M. C., Sizaret, P.-Y., Lamy, J., and van Bruggen, E. F. J. (1981) Biochemistry, 20, 1849-1856] has been refined. The relative positions in the half molecule of subunits Aa 3A and Aa 3B compared to those of Aa 3C and Aa 5B have been established by double labeling of the (2 X 6)-mer with binary mixtures of subunit-specific Fab fragments. The results show that subunits Aa 3B and Aa 5B are located in the same hexamer while Aa 3A and Aa 3C are in the other half of the (2 X 6)-mer. The choice of the enantiomer was deduced from a careful examination of electron micrographs of the native molecule. Finally a position was assigned to each of the 24 subunits on the flip and flop faces as defined by Van Heel and Frank [Ultramicroscopy, 6, 187-194 (1981)].

The extent and kinetics of reassembly of the four groups of linkers L1-L4 with 213 kDa subassemblies of twelve globin chains D, (bac)3(d)3, isolated from the approximately 3.6 MDa hexagonal bilayer (HBL) hemoglobin (Hb) of Lumbricus terrestris, was investigated using gel filtration. The reassembled HBL's were characterized by scanning transmission electron microscopic (STEM) mass mapping and their subunit content determined by reversed-phase chromatography. In reassembly by method (A), the linkers isolated by RP-HPLC at pH approximately 2.2 were added to D at neutral pH; in method (B), the linkers were renatured at neutral pH and then added to D. With method (A) the percentage of HBL reassembly varied from >/=13% in the absence of Ca(II) to /=75%), with ternary and binary linker combinations (40-50%) and with individual linkers producing yields increasing in the following order: L1=1-3%, L2 approximately L3=10-20% and L4=35-55%. The yield was two- to eightfold lower with method (B), except in the case of linkers L1-L3. Although the reassembly kinetics were always biphasic, with t1/2=0.3-3.3 hours and 10-480 hours, the ratio of the amplitudes fast:slow was 1:0.6 with method (A) and 1:2.5 with method (B). These results are consistent with a scheme in which the slow HBL reassembly is dependent on a slow conversion of linker conformation at neutral pH from a reassembly incompetent to a reassembly competent conformation. Although all the linkers self-associate extensively at neutral pH, forming complexes ranging from dimers to >18-mers, the size of the complex does not affect the extent or rate of reassembly. The oxygen binding affinity of reassembled HBLs was similar to that of the native Hb, but their cooperativity was lower. A model of HBL reassembly was proposed which postulates that binding of linker dimers to two of the three T subunits of D causes conformational alterations resulting in the formation of complementary binding sites which permit lateral self-association of D subassemblies, and thus dictate the formation of a hexagonal structure due to the 3-fold symmetry of D.

A 3D reconstruction of the hemoglobin (Hb) of the earthworm Lumbricus terrestris was carried out by the 3D projection alignment method from electron microscopy images of a frozen-hydrated specimen at 22 A resolution. The results were analyzed by a new approach taking into account the evolution of the 210 densities forming the 3D volume as a function of the threshold of surface representation. The whole oligomer with D6point-group symmetry is comprised of 12 hollow globular substructures (HGS) with local 3-fold symmetry tethered to a complex network of linking subunits (linker complex). The 12 globin subunits of each HGS are distributed around local 3-fold axis in four layers of three subunits. The first layer, the most external, contains monomeric globin chains 2A, 3A, and 5A. The three trimers corresponding to the nine remaining subunits have one subunit in each of the second (2B, 3B, 5B), third (1A, 4A, 6A), and fourth (1B, 4B, 6B) layer. The distances between the centers of the globin chains forming the trimers are in the ranges 20–32 A and 45–52 A. The linker complex is made up of two types of linking units. The first type forms three loops connecting globin chains of the second, third and fourth layers. The average molecular mass (Mm) of these subunits was 25 kDa. The second type forms the central structure, termed hexagonal toroid, and its 12 connections to the HGS. This structure corresponds to a hexamer of a single linking unit with a Mm (31.2 kDa), size and a shape different from those of the HGS loops. A careful study of 3D volume architecture shows that each toroid linking unit is bound to the three loops of a HGS pair located in the upper and lower hexagonal layers, respectively. As shown in a model of architecture, hexagonal bilayered (HBL) Hbs can be built very simply from 144 globin chains and 42 linker chains belonging to two different types. We also propose a simple assembly sequence for the construction of HBL Hbs based on the architecture model.

The refined three-dimensional structure of native human alpha2-macroglobulin (alpha2M) has been determined by cryoelectron microscopy and three-dimensional reconstruction. New features corresponding to "sigmoid arches," "basal bodies," and "apical connections" were observed in the molecule. Since similar elements are found in the architecture of transformed alpha2M, the 2 volumes were aligned in three dimensions. In their common orientations, they show many similarities except near the openings of the central chamber. In the native conformation, these apertures are fully opened, allowing the proteases to access the central chamber of the molecule, while in the transformed structure, they are partially closed. These structures suggest that alpha2M conformational change involves a strong lateral compression and a vertical stretching of the native particle seen in its four-petaled flower view to produce the H view of the transformed form. A model of structural transformation, in which all the parts of the alpha2M molecule seem involved in the entrapment of the proteinases is proposed.

Laboratoire des Proteines Complexes, CNRS URA 1334, Universite de Tours, 37032 Tours, France, Micromass UK Limited, 3 Tudor Road, Altrincham, Cheshire WA14 5RZ, UK, Equipe d’Ecophysiologie, Station Biologique, UPMC-CNRS-INSU, BP 74, 29682 Roscoff, France, Biology Department, Brookhaven National Laboratory, Upton, New York 11973, Department of Zoophysiology, Institute of Biological Sciences, Aarhus University, 8000 Aarhus C, Denmark, and Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, Michigan 48201

Cysteine 949 and glutamine 952 are known to be part of the thiol ester site of each of the four subunits of human alpha 2-macroglobulin (alpha 2M). The hydrolysis of this thiol ester bound to methylamine results in the incorporation of the amine and liberation of a free sulfhydryl group that can be specifically labeled. Therefore, a high-resolution marker specific for the sulfhydryl groups, the monomaleimido Nanogold (Au1.4nm) cluster was used to bind this amino acid. After cryoelectron microscopy, a three-dimensional reconstruction of the alpha 2M-Nanogold conjugates (alpha 2M-Au1.4nm) was achieved, revealing the internal location of the thiol ester sites in the transformed alpha 2M molecules. From this study we propose three possible locations for the cysteine 949.

A 3D reconstruction at 25 A resolution of native hemoglobin of the polychaete worm Arenicola marina was carried out from frozen-hydrated specimens examined in the electron microscope. The reconstruction volume of this large extracellular multimeric respiratory pigment appears as a hexagonal bilayer structure with eclipsed vertices in its upper and lower hexagonal layers. Conversely, in hemoglobins of oligochaetes, achaetes, and vestimentiferans and in chlorocruorins of the Sabellidae (polychaete) family, the vertices of the upper layer are 16 degrees clockwise rotated with respect to those of the lower layer. The fact that two other polychaete hemoglobins (Alvinella pompejana and Tylorrhynchus heterochaetus) have the same architecture as Arenicola led us to define two types of hexagonal bilayer hemoglobins/chlorocruorins: (i) type-I present in oligochaete, achaete, and vestimentiferan hemoglobins and in Sabellidae chlorocruorins; and (ii) type-II present in polychaete hemoglobins. A comparative study of the hemoglobins of Lumbricus terrestris (type-I) and Arenicola marina (type-II) showed that only two small differences located in the c4 and c5 linking units are responsible of the important architectural difference present in oligomers. A likely scheme proposed to explain the phylogenic distribution of the two types suggests that Clitellata, Sabellida (polychaete), and vestimentiferan hemoglobins and chlorocruorins derive from a type-I ancestral molecule, while Terebellida (Alvinella), Phyllodocida (Tylorrhynchus), and Scolecida (Arenicola) and possibly other polychaetes derive from an ancestor molecule with type-II hemoglobin. The architectures of the hollow globular substructures are highly similar in Arenicola and Lumbricus hemoglobins, with 12 globin chains and three linking units (c3a, c3b, and c4). The central piece of Arenicola hemoglobin is an ellipsoid while that of Lumbricus is a toroid. No phylogenic correlation could be found between the structure of the central pieces and the architecture type.

Hemocyanins, the respiratory molecules of cephalopod mollusks, are hollow cylinders with five internal arches. Three hemocyanins representative of three orders of cephalopods ( Benthoctopus species, Octopoda; Vampyroteuthis infernalis, Vampyromorpha; Sepia officinalis, Sepioidea) were subjected to cryoelectron microscopy and three-dimensional (3D) reconstruction. The structure of Benthoctopus hemocyanin, solved at 26.4-A resolution, possesses arches comprising two identical functional units. The similarity between these functional units and the structure recently observed in X-ray crystallography for Octopus by Cuff et al. ( J. Mol. Biol., 1998, 232, 522–529) allows the identification of their N- and C-terminal domains in the 3D reconstruction volume. Conversely, arches present in the 3D reconstruction volume of Sepia hemocyanin (21.8 A resolution) contain four functional units that are disposed differently. The strong resemblance between the reconstruction volumes of Vampyroteuthis (21.4-A resolution) and Benthoctopus hemocyanins suggests that Sepioidea diverged from a group containing Octopoda and Vampyromorpha.

Hemocyanin (Hc) the respiratory pigment of the horseshoe crab Limulus polyphemus (Lp) is composed of 48 ∼75 kDa copper-containing subunits arranged in eight hexameric groups. In this study, we used the random conical tilt series method to do a three-dimensional (3D) reconstruction of Lp Hc observed in vitreous ice. This approach allowed the unambiguous determination of the handedness of the molecule. Lp Hc contains two superimposed 4 × 6mer structures possessing the same structural features as the other 4 × 6meric Hcs, namely flip and flop views and a rocking effect. Moreover, 3D fitting of the X-ray structure of subunit Lp II with the reconstruction volume shows that the intra 4 × 6meric contacts described in arthropod Hcs also occur within Limulus Hc. The two half-molecules composing the 8 × 6mer have their flop faces in contact (flop/flop association), the main links being formed by subunits Lp IV. Model building shows that the flop/flop association is the only possible arrangement which allows the assembly of the whole particle. The two alternate constructions (flip/flop and flip/flip) are forbidden because of steric hindrance.

Laboratoire des Proteines Complexes, CNRS URA 1334, Universite de Tours, 37032 Tours, France, Micromass UK Limited, 3 Tudor Road, Altrincham, Cheshire WA14 5RZ, UK, Equipe d’Ecophysiologie, Station Biologique, UPMC-CNRS-INSU, BP 74, 29682 Roscoff, France, Biology Department, Brookhaven National Laboratory, Upton, New York 11973, Department of Zoophysiology, Institute of Biological Sciences, Aarhus University, 8000 Aarhus C, Denmark, and Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, Michigan 48201

An immunocomplex formed by the 4 × 6 meric hemocyanin of the scorpion Androctonus australis with the monoclonal antibody L104 was studied by cryoelectron microscopy and subjected to three-dimensional (3D) reconstruction. The reconstructed particle reflects the structure of the immunocomplex in its hydrated state and is devoid of the flattening that was previously observed with a negatively stained preparation. A 3D fitting of the X-ray data of the Panulirus interruptus hemocyanin and of the Fab R19.9 to the reconstruction volume allowed the first quantitative measurement of the structural parameters of the antigen, and the localization of the epitope at the surface of subunit Aa 6. The independent alignment of the Fobs and the hexamers provides a direct verification for the accuracy of the fit and allows the building of the most detailed model of a cheliceratan 4 × 6meric complex and its attached monoclonal antibodies.

A frozen-hydrated sample embedded in vitreous ice of human α2-macroglobulin transformed by methylamine was imaged by cryoelectron microscopy and reconstructed in three dimensions. In the reconstruction, the cage-like architecture of this protease inhibitor is fully revealed with a clear visualization of two lozenge-shaped lateral walls connected by thin bridges. The shape and dimensions of the internal cavity normally containing the trapped protease(s) is described. The possible locations of the thiol ester sites and intersubunit connections are also discussed.

Arthropod Hcs have highly heterogeneous Mws ranging from approximately 450 to 3,600 kDa. For example, lx6-mers of 75 kDa polypeptide chains have been found in crustaceans, 2x6-mers in crustaceans and spiders, 4x6-mers in most chelicerate groups and in the thalassinid shrimps, 6x6-mers in centipedes, and 8x6-mers in merostoms.

Comparative x-ray scattering experiments and electron microscopic observations have been performed on native S-form, and on different F-forms of human plasma α2-macroglobulin (α2M), obtained by proteinase (chymotrypsin, plasmin, and thrombin) or methylamine treatment. Image processing of electron micrographs of the α2M molecules transformed by chymotrypsin, plasmin, and methylamine displayed average images which could be compared. The proteinase-complex α2M molecules exhibited the usual H-like structure, but the methylamine-inactivated ones showed a different organization, with almost no stain-excluding material in the central region of the molecule, which therefore presented a central cavity filled with stain. By subtracting average images of α2M-methylamine from α2M-chymotrypsin or α2M-plasmin, a putative localization of the proteinases inside the α2M molecule, very close to its center was revealed. The values of the radii of gyration for the S- and F-forms obtained by x-ray scattering were very different (78 and 67.7 A, respectively). All four scattering curves of the F-forms were comparable in shape and showed maxima and minima different from that of the S-form α2M. Image processing of electron micrographs and x-ray scattering have provided independent results which indicate that a large cavity exists in the α2M-methylamine molecule and that the proteinases might be located in a very central position inside the α2M-proteinase molecules.

Immunological cross-reactivities between isolated subunits of the scorpion Androctonus australis (Aa) and of the horseshoe crab Limulus polyphemus (Lp) hemocyanins were studied using subunit-specific antibodies prepared through immunoadsorption to pure immobilized subunits. Rocket immunoelectrophoreses of the various subunits of both hemocyanins were carried out at constant antigen concentration against the various subunit-specific antibody preparations. Then the data were analyzed through factorial correspondence analysis and compared to the respective intramolecular locations of the subunits in both hemocyanins. The results show that the dimeric subunits located in the central part of each (4 × 6)meric structure ( Aa whole molecule and Lp half molecule) were strongly preserved. In addition, the (8 × 6)mer-forming subunit of Lp hemocyanin ( Lp IV) and the subunit occupying the same intramolecular position in Aa hemocyanin ( Aa 5A) were also strongly preserved. Besides the strong antigenic relatedness, less pronounced crossed immunoprecipitations or no precipitation at all were observed between subunits with homologous positions suggesting a minor structural and/or functional roles for these subunits. All the antigen-antibody combinations leading to an absence of immunoprecipitation were screened for the presence of soluble immunocomplexes by radioimmunological tests. In all cases, soluble immunocomplexes were observed. These results suggest the following evolution scenario. First, the central dimeric subunits, responsible of the dodecamer aggregation ( Aa 3C and 5B and Lp V and VI) were already differentiated when Merostomata diverged from Arachnida. Second, the differentiation of the (8 × 6)mer-forming subunit occurred in the Merostomata ramification in a preserved subunit already possessing a functional advantage. Third, the differentiation of subunits Aa 3A and Aa 3E recently occurred in the scorpion ramification.

The hemocyanin of Octopus dofleini has provided an excellent model for the study of the behavior of multi-subunit proteins (1–3). Sedimentation experiments indicated that the native molecule (Mr = 3.6 × 106, S20,w =51S) is a decamer of polypeptide chains (Mr = 3.6 × 105, S20,w = 11.1S). Preliminary electrophoretic studies suggested that there might be only a single kind of polypeptide chain, making this structure simpler than that of most molluscan hemocyanins. Furthermore, the molecular weight of the subunit is smaller than that of gastropod hemocyanins, suggesting that it might contain fewer than eight oxygen binding domains. We report here on our investigations of the structure of O. dofleini hemocyanin.

Translation of Androctonus australis poly(A)-RNA in vitro led to a number of polypeptides products (8–10) of 70–73 kDa analyzed by two-dimensional gel electrophoresis and identified by immunoprecipitation with an anti- (dissociated hemocyanin) antiserum. The translated hemocyanin polypeptides have the same physico-chemical characteristics as authentic hemocyanin subunits. Subunits Aa 2 and Aa 4 have been identified with monospecific antisera characterized (a) by their capability of reacting with their homologous subunit and (b) by their inability of binding to cross-reacting subunits. Each polypeptide chain is coded by a different messenger without significant post-translational events. Hemocyanin could be detected among the translation products of the poly(A)-RNA isolated from the cuticle under the carapace.