Erythrocruorin from the water-flea Daphnia magna. Quaternary structure and arrangement of subunits.

The subunit structure of erythrocruorin from the cladoceran Daphnia magna was studied. The native protein was found to have a sedimentation coefficient (S2(20), w) of 17.9 +/- 0.2 S and a molecular weight, as determined by sedimentation equilibrium, of 494 000 +/- 33 000. Iron and haem determinations gave 0.312 +/- 0.011% and 3.84 +/- 0.04%, corresponding to minimal molecular weights of 17900 +/- 600 and 16 100 +/- 200 respectively. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis gave one band with mobility corresponding to a molecular weight of 31 000 +/- 1 500. The molecular weight of the polypeptide chain determined by sedimentation equilibrium in 6 M-guanidinium chloride and 0.1 M-2-mercaptoethanol is 31 100 +/- 1300. On a molecular-weight basis, Daphnia erythrocruorin is composed of 16 identical polypeptide chains carrying two haem groups each. The native structure is stable between pH5 and 8.5. At alkaline and acidic pH, a gradual decrease in the sedimentation coefficient down to 9.8S occurs. Above pH 10 and below pH4, a slow component with S20, w between 2.7S and 4.0S is observed. The 2.7S, 4.0S and 9.8S species are identified as single-chain subunits, subunit dimers and half-molecules respectively. We propose a model for the molecule composed of 16 2.7S subunits grouped in two layers stacked in an eclipsed orientation, the eight subunits of each layer occupying the vertices of a regular eight-sided polygon. Support for this arrangement is provided from electron microscopy and from analysis of the pH-dissociation pattern.     

Electron microscopy and image analysis of the GroEL-like protein and its complexes with glutamine synthetase from pea leaves.

The molecular structure of GroEL-like protein from pea leaves has been studied by electron microscopy and image analysis of negatively stained particles. Over 1500 molecular projections were selected and classified by multivariate statistical analysis. It was shown that the molecule consists of 14 subunits arranged in two layers with 72 point group symmetry. Side view projections of the molecule show a four-striation appearance, which subdivides both layers of seven subunits into two halves; this may be explained by a two-domain structure of the subunits. The presence in protein preparations of projections corresponding to one layer of subunits or half-molecules is consistent with the molecular structure suggested. Electron microscopic evidence for a specific association of GroEL-like protein and octameric glutamine synthetase, which was co-purified with this protein, was obtained.     

Interaction between Protein Subunits from Model Studies

A molecular model of hemoglobin was constructed which made it possible to visualize the relation between various amino acid residues in the molecule. The model indicates that electrostatic forces might play a significant role in holding the subunits of hemoglobin together. This would explain why myoglobin does not form a tetramer while four β-chains, which are structurally similar to myoglobin, do assemble into a hemoglobin H molecule. Also, as far as the primary structures of hemoglobin chains of various species are known, the proposed ionic links between subunits are consistent with the fact that mammalian hemoglobins form stable tetramers while the peptide chains of lamprey hemoglobin are only weakly associated. The different behavior of hemoglobin H and of normal hemoglobin upon oxygen uptake is briefly discussed in terms of allosteric effects.     

Structure of the polypeptide chain of extracellular hemoglobin from the nematode Ascaris suum.

1. Ascaris suum extracellular hemoglobin is composed of eight identical single polypeptide chain subunits carrying two heme binding sites each. 2. Limited trypsinolysis followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis gave a major band corresponding to half the molecular mass of an intact subunit. 3. Peptide mapping of tryptic hydrolysates yielded 27 to 30 fluorescamine positive spots, about half the number of lysyl and arginyl residues in a polypeptide chain. 4. The findings indicate that a subunit of Ascaris hemoglobin consists of two structural units of roughly equal size, corresponding to two recurring sequences, connected together by the continuity of the polypeptide chain.     

A re-evaluation of the molecular mass of earthworm extracellular hemoglobin from meniscus depletion sedimentation equilibrium. Nature of the 10 S dissociation species.

Previous calculations from meniscus depletion sedimentation equilibrium earthworm hemoglobin from Lumbricus terrestris (E.J. Wood et al., Biochem. J. 153 (1976) 589-96) and from the related species Lumbricus sp. (L. sp.) (M.M. David and E. D Mol. Biol. 87 (1974) 89--101) were made on the assumption that the solutions behaved ideally. Re-examination of their results reveals, however, a dependence of the apparent molecular mass on concentration. Taking this effect into consideration, we have nowrecalculated from their data molecular masses of 4.4--4.5 MDa for the hemoglobin of both L. terrestris and L. sp. On the basis of the new determinations, we propose for the polypeptide chain composition of L. terrestris hemoglobin a model [(abcd )4L1L2L3]12 where a,b,c,d are the four globin and L1,L2,L3 are the three major linker chain constituents of the protein. The model is consistent with the D6 symmetry of the molecule. A 10 S intermediate product in the alkaline dissociation Lumbricus hemoglobin is viewed as a binary mixture of products resulting from a disproportionation reaction involving the structural unit. The present interpretation is shown to be consistent with observed relations between molecular masses and SDS gel electrophoretic band patterns of 10 S species and intact hemoglobin.     

Subunit arrangement of Escherichia coli F1-ATPase studied by scanning transmission electron microscopy

The shape and the arrangement of subunits in Escherichia coli F1-ATPase (ECF1) lacking the delta subunit have been explored with a high performance scanning transmission electron microscope. In tilting experiments, the ECF1 molecule appeared as a flat cylinder whose width (approx. 120 A) was about twice its height. The symmetry of front view projections of ECF1 has been investigated by computer analysis. In a population taken at random from the data bank, one third of the particles showed five-fold radial symmetry components, one third six-fold radial symmetry components and the last third no typical symmetry. The six-fold radial symmetry was consistent with a hexagonal arrangement of six large peripheric masses, which probably correspond to the three alpha and the three beta subunits of ECF1. The five-fold radial symmetry was tentatively explained by a fusion of two juxtaposed peripheric subunits. Lateral projections showed a zig-zag organization of the large masses, suggesting that the large alpha and beta subunits are located on two levels, with some degree of intercalation between the subunits of the two levels.     

Low resolution crystal structure of Arenicola erythrocruorin: influence of coiled coils on the architecture of a megadalton respiratory protein

Annelid erythrocruorins are extracellular respiratory complexes assembled from 180 subunits into hexagonal bilayers. Cryo-electron microscopic experiments have identified two different architectural classes. In one, designated type I, the vertices of the two hexagonal layers are partially staggered, with one hexagonal layer rotated by about 16 degrees relative to the other layer, whereas in the other class, termed type II, the vertices are essentially eclipsed. We report here the first crystal structure of a type II erythrocruorin, that from Arenicola marina, at 6.2 A resolution. The structure reveals the presence of long continuous triple-stranded coiled-coil "spokes" projecting towards the molecular center from each one-twelfth unit; interdigitation of these spokes provides the only contacts between the two hexagonal layers of the complex. This arrangement contrasts with that of a type I erythrocruorin from Lumbricus terrestris in which the spokes are broken into two triple-stranded coiled coils with a disjointed connection. The disjointed connection allows formation of a more compact structure in the type I architecture, with the two hexagonal layers closer together and additional extensive contacts between the layers. Comparison of sequences of the coiled-coil regions of various linker subunits shows that the linker subunits from type II erythrocruorins possess continuous heptad repeats, whereas a sequence gap places these repeats out of register in the type I linker subunits, consistent with a disjointed coiled-coil arrangement.     

Assembly of the regulatory complex of the 26S proteasome

The 19S regulatory complex (RC) of 26S proteasomes is a 900–1000 kDa particle composed of 18 distinct subunits (S1–S15) ranging in molecular mass from 25 to 110 kDa. This particle confers ATP-dependence and polyubiquitin (polyUb) recognition to the 26S proteasome. The symmetry and homogenous structure of the proteasome contrasts sharply with the remarkable complexity of the RC. Despite the fact that the primary sequences of all the subunits are now known, insight has been gained into the function of only eight subunits. The six ATPases within the RC constitute a subfamily (S4-like ATPases) within the AAA superfamily and we have shown that they form specific pairs in vitro[1]. We have now determined that putative coiled-coils within the variable N-terminal regions of these proteins are likely to function as recognition elements that direct the proper placement of the ATPases within the RC. We have also begun mapping putative interactions between non-ATPase subunits and S4-like ATPases. These studies have allowed us to build a model for the specific arrangement of 9 subunits within the human regulatory complex. This model agrees with recent findings by Glickman et al. [2] who have reported that two subcomplexes, termed the base and the lid, form the RC of budding yeast 26S proteasomes.     

The structural features of beef heart mitochondrial creatine kinase.

Two forms of mitochondrial creatine kinase (Mi-CK) having Mr 320 kDa and 240 kDa as determined by gel-filtration on Sephacryl S-300 in 0.1 M Tris-HCl pH 7.4 were investigated. The sedimentation coefficient values for these two forms were found to be identical and equal to 12.3 S. When studied by electron microscopy the main type of images for the 320 kDa and 240 kDa Mi-CK appeared as annular particles, 12-14 nm in diameter, with a well-detected subunit structure and a central hollow, 3-4nm in diameter filled with the dye. The results of the averaging of the main type of individual Mi-CK images and particles of the two-dimensional crystal layer point to the overall geometry of the Mi-CK molecule structure as containing eight subunits arranged by a 4-fold symmetry around the central hollow. It may be that the eight identical subunits of crystalline Mi-CK are arranged with a P422 symmetry. However in both cases the averaged main images do not show a mirror symmetry. The multiplicity of the observed projections close to annular one provides additional evidence in favour of the great lability and structural mobility of the Mi-CK subunits. It allows to assume that two forms (320 kDa and 240 kDa) are not the different oligomers but they are two functionally distinct conformational states of octameric molecule of Mi-CK.     

Three-dimensional structure of the surface layer protein of Aquaspirillum serpens VHA determined by electron crystallography.

The three-dimensional structure of the protein which forms the S layer of Aquaspirillum serpens strain VHA has been determined by electron microscopy. Structures have been reconstructed to a resolution of about 1.6 nm for single-layered specimens and about 4 nm for two-layered specimens. The structure, which has hexagonal symmetry, consists of a core in the shape of a cup, with six projections arising from the rim of the cup to join adjacent subunits at the threefold symmetry axes. The model is consistent with edge views of the S layer which have been obtained in this and other work. It is now clear from this work and from three-dimensional reconstructions of other bacterial S layers that a wide diversity exists in the morphology of surface layers.     

Purification and properties of alcohol oxidase from <Emphasis Type="Italic">Pichia putida</Emphasis>

Alcohol oxidase (AO) was extracted from the methylotrophic yeast Pichia putida and purified using various methods. AO purified by crystallization was homogeneous based on analytical centrifugation with subsequent gel filtration and SDS-PAGE. The molecular weight of the enzyme was around 600 kDa. SDS-PAGE revealed a single protein band (74 ± 4 kDa), and 8–9 bands of native protein with similar specific AO activities and substrate specificities were identified by PAGE without SDS. Electron microscopy of a single molecule revealed eight subunits located on the top of a regular tetragon with dotted symmetry of 422D4 providing evidence that AO consists of eight subunits. Apparently, each molecule of AO has two types of subunits with very similar molecular weights and differing from each other by the number of acidic and basic amino acid residues. Each subunit includes one molecule of FAD and 2–3 cysteine residues. The pH optimum was within 8.5–9.0. Specific activity of the enzyme varied from 10 to 50 μmol methanol/min per mg protein from batch to batch depending on separation methods and had linear relationship with protein concentration. The AO was quickly inactivated at 20°C and seemed to be stable in phosphate-citrate buffer with 30–50% (w/v) of sucrose. Different forms of 0.1–1 mm crystals of the enzyme were obtained. However the crystals did not yield X-ray reflections, apparently as a result of their molecular microheterogeneity.     

The structure of form I crystals of D-ribulose-1,5-diphosphate carboxylase.

Single crystals of d-ribulose-1,5-diphosphate carboxylase from tobacco leaves, Nicotiana tabacum (variety Turkish Samsun), have been examined by X-ray diffraction, electron microscopy, and optical diffraction. Twelve molecules are loosely packed into a body-centered cubic unit cell, space group I4¹32 with cell dimension $\text{a = 383}\text{A}\text{̊}$. The asymmetric unit is one quarter of a molecule, and the minimum molecular symmetry is 222. This symmetry when combined with estimates of the two subunit masses and stoichiometry is compatible with a molecular structure of the composition L₈S₈ (L is large subunit, S is small). If all bonds between large and small subunits are equivalent, the true molecular symmetry is 422; this symmetry is consistent with molecular images in micrographs.     

An electron-microscope study of beta-glucuronidase crystals.

beta-Glucuronidase from rat preputial glands was crystallized as thin sheets having p6 symmetry in projection with a equal 20.2nm. A filtered image was produced by Fourier methods to a resolution of 2.2 nm by averaging information from six areas. This suggests an approximately triangular molecular outline in projection, and this is taken to indicate a probable tetrahedral arrangement of the four subunits of the beta-glucuronidase molecule.     

Molecular mass of macromolecules and subunits and the quaternary structure of hemoglobin from the microcrustacean Daphnia magna

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.     

pH-dependent conformational transformation in mung bean glyceraldehyde-3-phosphate dehydrogenase

In thermal inactivation at pH 7.3 and below, the tetrameric apo-glyceraldehyde-3-phosphate dehydrogenase of mung beans lost half of its activity more rapidly than the rest, suggesting a pairwise arrangement of subunits (or a C2 symmetry). At pH 8.6, the activity was lost in a single exponential decay, characteristic of functional identity of sites as in a tetrahedral arrangement of subunits (or a D2-type symmetry). At intermittent pH values, the kinetics of thermal inactivation were consistent with the presence of a mixture of C2- and D2-symmetry conformations. In "sudden pH change" experiments, the observed thermal inactivation kinetics were characteristic of the final pH at which the enzyme was heated. Thus, the interconversion of the two conformations is facile and very fast. There is no gross change in molecular weight of the enzyme between pH 7.3 and 8.6.     

Three-dimensional reconstruction of Lumbricus terrestris hemoglobin at 22 A resolution: intramolecular localization of the globin and linker chains.

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.     

Small angle x-ray scattering of the hemoglobin from Biomphalaria glabrata

The hemoglobin from Biomphalaria glabrata is an extracellular respiratory protein of high molecular mass composed by subunits of 360 kDa, each one containing two 180 kDa chains linked by disulfide bridges. In this work, small angle x-ray scattering (SAXS) measurements were performed with the hemoglobin at pH 5.0 and 7.5. Radii of gyration of 98.6 +/- 0.5 and 101.8 +/- 0.2 A and maximum diameters of 300 +/- 10 and 305 +/- 10 A, respectively, were obtained from Guinier plot extrapolation and analytical curve fitting. The pair distance distribution functions p(r) corresponded to globular particles with a somewhat anisotropic shape for both preparations. Computer analysis of the low angle part of the scattering curve led to the determination of the low resolution envelope of the protein, revealing a P(222) symmetry. Shape reconstruction from ab initio calculations using the complete scattering curve furnished a compact prolate three-dimensional (3D) bead model for the protein. Hydrodynamic parameters were obtained from experiments and theoretical calculations using the 3D model. The results of the structural and biochemical studies reported herein indicate that the multisubunit structure of this hemoglobin is compatible with a tetrameric arrangement.     

Modeling of possible subunit arrangements in the eukaryotic chaperonin TRiC

The eukaryotic cytosolic chaperonin TRiC (TCP-1 Ring Complex), also known as CCT (Cytosolic Chaperonin containing TCP-1), is a hetero-oligomeric complex consisting of two back-to-back rings of eight different subunits each. The general architecture of the complex has been determined, but the arrangement of the subunits within the complex remains an open question. By assuming that the subunits have a defined arrangement within each ring, we constructed a simple model of TRiC that analyzes the possible arrangements of individual subunits in the complex. By applying the model to existing data, we find that there are only four subunit arrangements consistent with previous observations. Our analysis provides a framework for the interpretation and design of experiments to elucidate the quaternary structure of TRiC/CCT. This in turn will aid in the understanding of substrate binding and allosteric properties of this chaperonin.