Identification of two antibody-interaction sites on the surface of Panulirus interruptus hemocyanin.

Negatively stained complexes of Panulirus interruptus (spiny lobster) hemocyanin with two different monoclonal antibodies, named E and J, were studied by electron microscopy and image processing. The attachment site of the antibodies to the hexameric hemocyanin molecule was deduced from two perpendicular views of hemocyanin/antibody complexes, in which either the threefold axis or one of the twofold axes was oriented perpendicular to the supporting film. Images of complexes in these orientations were searched with reference images simulated from the known X-ray structure of P. interruptus hemocyanin. The two sites were further characterized by combining our results from electron microscopy with structural data obtained by X-ray diffraction and other methods. These two antibodies recognize different non-overlapping epitopes. The epitope for clone E is located on domain 3 at the surface of the beta barrel and consists of certain loops, which form connections between beta-strand structures. The epitope for clone J is situated on domain 1 at the surface of an alpha-helical region and consists mainly of certain alpha-helices connecting loops. The orientation of the hemocyanin hexamers in the two complexes is very different, as is demonstrated most clearly when they form chains. Clone E forms complexes with the threefold axes perpendicular to the chain direction, while for clone J the threefold axes seem to be parallel to the main direction. The angle between the Fab part of an IgG molecule and the threefold axis of the hexamer is 60 +/- 5 degrees for clone E and 35 +/- 7 degrees for clone J. This observation is clearly related to the difference in orientation of the hexamers for the two complexes.

The interhexameric contacts in the four-hexameric hemocyanin from the tarantula Eurypelma californicum. A tentative mechanism for cooperative behavior.

Arthropod hemocyanins (Hcs) are regular assemblies of 1, 2, 4, 6 or 8 hexameric protein molecules. They transport oxygen and can bind it in a cooperative manner. The hexameric X-ray structures of Panulirus interruptus (spiny lobster) and of Limulus polyphemus (horseshoe crab) subunit II Hc were solved recently by the groups of Hol (Groningen, The Netherlands) and Magnus (Cleveland, U.S.A.). They related cooperativity to a rotational movement of a domain within a subunit and of the two trimers mutually inside the hexamer. In our study a model was derived for the structure and related to the function of the four-hexameric Hc from the tarantula Eurypelma californicum by combining data from electron microscopy and image processing, from the X-ray diffraction studies mentioned earlier and from amino acid sequence studies. Interhexameric contacts were determined at the level of secondary structure elements and in some cases of single amino acids. Loops, undefined in the X-ray structures of the hexamers, were often involved in these contacts. In one case the contact was formed between four parallel alpha-helices, two from each hexamer. Based on these findings a mechanism is proposed for the transmission of cooperativity between the hexamers, in which the concept of "helical friction" plays a key role.

Purification and characterization of an oxygen-labile, NAD-dependent alcohol dehydrogenase from Desulfovibrio gigas.

A NAD-dependent, oxygen-labile alcohol dehydrogenase was purified from Desulfovibrio gigas. It was decameric, with subunits of M(r) 43,000. The best substrates were ethanol (Km, 0.15 mM) and 1-propanol (Km, 0.28 mM). N-terminal amino acid sequence analysis showed that the enzyme belongs to the same family of alcohol dehydrogenases as Zymomonas mobilis ADH2 and Bacillus methanolicus MDH.

Electron microscopic analysis and structural characterization of novel NADP(H)-containing methanol: N,N'-dimethyl-4-nitrosoaniline oxidoreductases from the gram-positive methylotrophic bacteria Amycolatopsis methanolica and Mycobacterium gastri MB19.

The quaternary protein structure of two methanol:N,N'-dimethyl-4-nitrosoaniline (NDMA) oxidoreductases purified from Amycolatopsis methanolica and Mycobacterium gastri MB19 was analyzed by electron microscopy and image processing. The enzymes are decameric proteins (displaying fivefold symmetry) with estimated molecular masses of 490 to 500 kDa based on their subunit molecular masses of 49 to 50 kDa. Both methanol:NDMA oxidoreductases possess a tightly but noncovalently bound NADP(H) cofactor at an NADPH-to-subunit molar ratio of 0.7. These cofactors are redox active toward alcohol and aldehyde substrates. Both enzymes contain significant amounts of Zn2+ and Mg2+ ions. The primary amino acid sequences of the A. methanolica and M. gastri MB19 methanol:NDMA oxidoreductases share a high degree of identity, as indicated by N-terminal sequence analysis (63% identity among the first 27 N-terminal amino acids), internal peptide sequence analysis, and overall amino acid composition. The amino acid sequence analysis also revealed significant similarity to a decameric methanol dehydrogenase of Bacillus methanolicus C1.

Architecture of peroxisomal alcohol oxidase crystals from the methylotrophic yeast Hansenula polymorpha as deduced by electron microscopy.

The architecture of alcohol oxidase crystalloids occurring in vivo in the peroxisomes of methylotrophic yeasts was deduced from electron micrographs of similar crystals of the Hansenula polymorpha enzyme grown in vitro. Three characteristic views of the crystal are observed, as well as single layers in the very early stages of crystal formation. The crystal is concluded to be cubical, with every octameric molecule making the same contacts with four neighbors in one plane, at right angles to its fourfold axis. The unit cell contains six octamers, in three mutually orthogonal orientations, and two large holes, which can accommodate other peroxisomal proteins involved in methanol metabolism. The crystal contains channels, connecting the holes, which allow the diffusion of relatively large molecules through the crystal. Crystal formation depends on just one contact per subunit, which may explain the fragility of the crystals.

Electron microscopic analysis and biochemical characterization of a novel methanol dehydrogenase from the thermotolerant Bacillus sp. C1.

Methanol dehydrogenase from the thermotolerant Bacillus sp. C1 was studied by electron microscopy and image processing. Two main projections can be distinguished: one exhibits 5-fold symmetry and has a diameter of 15 nm, the other is rectangular with sides of 15 and 9 nm. Subsequent image processing showed that the 5-fold view possesses mirror symmetry. The rectangular views can be divided into two separate classes, one of which has 2-fold rotational symmetry. It is concluded that methanol dehydrogenase is a decameric molecule, and a tentative model is presented. The estimated molecular weight is 430,000, based on a subunit molecular weight of 43,000. The enzyme contains one zinc and one to two magnesium ions per subunit. N-terminal amino acid sequence analysis revealed substantial similarity with alcohol dehydrogenases from Saccharomyces cerevisiae, Zymomonas mobilis, Clostridium acetobutylicum, and Escherichia coli, which contain iron or zinc but no magnesium. In view of the aberrant structural and kinetic properties, it is proposed to distinguish the enzyme from common alcohol dehydrogenases (EC 1.1.1.1) by using the name NAD-dependent methanol dehydrogenase.

Electron microscopy and image analysis of two-dimensional crystals and single molecules of alcohol oxidase from Hansenula polymorpha.

The octameric protein alcohol oxidase from the yeast Hansenula polymorpha was studied by electron microscopy and image analysis. Two-dimensional crystals were formed by applying the protein, in a phosphate buffer containing poly(ethylene glycol) and EDTA, to a carbon-coated formvar film which had been glow-discharged in pentylamine at least several hours earlier. The crystals show p4 symmetry and have a unit cell of 12.5 X 12.5 nm2, containing one molecule. Image analysis of the crystals and of single molecules yielded two different views. From these it can be deduced that the subunits have an elongated shape and form two layers of four, stacked face to face. A tentative model of the structure is presented.

Crystallization properties and structure of Panulirus interruptus haemocyanin.

Electron-microscopic studies revealed that two types of subunits of Panulirus interruptus haemocyanin crystallize in different ways. Homohexamers of subunit a give close-packed two-dimensional crystals whereas homohexamers of subunit c form open two-dimensional arrays. We applied computer-image analysis to these arrays and studied the differences in crystallization properties by combining the electron-microscopic data with amino acid sequence information and the X-ray diffraction model of subunit a.

Computer image analysis of two-dimensional crystals of beef heart NADH: ubiquinone oxidoreductase fragments. I. Comparison of crystal structures in various negative stains.

We investigated the structure of two-dimensional crystals from bovine heart mitochondrial NADH: ubiquinone oxidoreductase. A detailed description of uranyl acetate-stained crystals demonstrated that they are composed of fragments in a spatial arrangement according to space group P4212 [J. Brink, S. Hovmöller, C.I. Ragan, M.W.J. Cleeter, E.J. Boekema and E.F.J. van Bruggen, European J. Biochem. 166 (1987) 287]. To gain more structural information on the crystal structure and to assess the effects of various negative stains on the structure preservation and appearance, we examined stained crystals by means of electron microscopy and image analysis. The space group P4212 appeared to be present for several stains tested, i.e. ammonium molybdate, uranyl acetate, uranyl nitrate and uranyl sulphate. Use of phosphotungstic acid and silicotungstate resulted in a reduction of symmetry to pseudo-P4212 or p4. Use of sodium tungstate led to a considerable loss of resolution to 3.8 nm at best, whereas otherwise 1.5 to 1.9 nm could be demonstrated. The lattice vectors were not affected by the stains; they were determined as a = b = 14.9 +/- 0.25 nm with gamma = 89.8 degrees +/- 0.6 degrees. Image analysis showed the presence of similar structures with the molybdate and uranyl compounds. Differences were observed in the case of the tungstate type of stains. Furthermore, the analysis revealed the complete absence of the four small pores of 2.0 nm diameter in the unit cell. This effect was observed irrespective of the type of stain and supporting film, and could be ascribed only to the glow-discharge treatment of the supporting film. The observed difference must be caused by changed interactions between the protein, stain and supporting film. Application of correspondence analysis and clustering algorithms to the various reconstructed images of the crystals showed that they could be separated into several clusters. Each of these clusters corresponded on the average to only one type of stain, whereas a further division according to the specific uranyl compounds was observed. This study therefore shows that under identical preparation conditions subtle differences between individual stains can be detected.

Computer image analysis of two-dimensional crystals of beef heart NADH: ubiquinone oxidoreductase fragments. II. Comparison of frozen hydrated and negatively stained specimens.

Two-dimensional crystals of bovine NADH: ubiquinone oxidoreductase fragments were studied by cryo-electron microscopy and computer image analysis at two focus settings. The lattice parameters of the crystals were determined as a = 14.5 +/- 0.2 nm, b = 14.8 +/- 0.3 nm with gamma = 87.8 degrees +/- 0.2 degrees. Translation and rotation alignment resulted in a final resolution of 1.38 (close-to-focus) or 1.9 nm (1.3 microns underfocus). When only translational alignment was employed 1.6 nm resolution resulted (close-to-focus case). The application of a further correction procedure surprisingly lowered the achieved resolution to 2.2 nm. A combination of the projected structures for the two focus settings in Fourier space resulted in a noise-free image displaying enhanced image contrast for reciprocal spacings from 1/5.0 to 1/1.4 nm-1. The structure in amorphous ice comprises four separate quasi-identical motifs, each resembling a distorted triangle. Two symmetry-related (p2) motifs reveal a pore-like feature. In negative stain the structure has p4mm symmetry. The absence of in-plane symmetry elements (i.e. twofold rotation and screw axes) and other differences might be accounted for (at least in part) if the crystal studied was slightly tilted.

Electron microscopy and image analysis of the complexes I and V of the mitochondrial respiratory chain.

The results of Section IV can be summarized in a simple ATP synthase model. This model implies that either the alpha or the beta subunits must be closer to the membrane. The work of Gao and Bauerlein (1987) indicates that the alpha subunits are closer to the membrane. Although the overall structure is more or less clear, important questions need to be clarified. First, the number and the arrangement of the subunits in the F0 part must be known. Second, the exact shape of F1, and particularly the shape of the large subunits needs to be elucidated. On the basis of fluorescence resonance energy transfer measurements by McCarty and Hammes (1987), a model was presented showing large oblong subunits. Such 'banana-shaped' subunits, which are also presented in the many phantasy models (e.g. Walker et al., 1982), are very unlikely in view of the electron microscopical results, although the large subunits do not need to be exactly spherical. The third and most interesting central question is on the changes in the structure that take place during the different steps in the synthesis of ATP. It can now be taken as proven that the energy transmitted to the ATP synthase is used to induce a conformational change in the latter enzyme, in such a way as to bring about the energy-requiring dissociation of already synthesized ATP (Penefsky, 1985 and reviewed in Slater, 1987). But the way in which the three parts of the ATP synthase are involved is completely unknown. It is rather puzzling that such a long distance exists between the catalytic sites, which are on the interface of the alpha and beta subunits and the F0 part where the proton movements occur, which, according to Mitchell's theory (1961), is the driving force for the synthesis of ATP. Perhaps alternative mechanisms such as the collision hypothesis formulated by Herweijer et al. (1985) are more realistic in describing the mechanism of ATP synthesis. It would bring the complexes I and V close together, not only in the artificial way treated in this paper, but in a useful way for energy conversion.

The structure of NADH:ubiquinone oxidoreductase from beef-heart mitochondria. Crystals containing an octameric arrangement of iron-sulphur protein fragments.

We have investigated the structure of two-dimensional crystals from preparations of NADH:ubiquinone oxidoreductase from beef-heart mitochondria. The crystal structure of these crystals was previously determined to be equivalent with two native enzyme molecules per unit cell, i.e. a p2 symmetry [Boekema, E. J., Van Heel, M. G. & Van Bruggen, E. F. J. (1984) Biochim. Biophys. Acta 787, 19-26]. However, the optical diffraction patterns of the crystals displayed a clear fourfold symmetry. A Fourier analysis carried out on the calculated diffraction pattern proved unambiguously that the crystal symmetry was p42(1)2. Following crystallographic rules the unit cell therefore contained eight identical molecules. As a consequence, only a subcomplex of the enzyme rather than the intact enzyme formed the crystal. Electron microscopy of isolated, single molecules of the iron-sulphur protein, a dissociation product of complex I, revealed the presence of square complexes with sides of approximately 15 nm. Since these complexes were indistinguishable from the building blocks (unit cells) of the two-dimensional crystals, the crystals could be composed of Fe-S protein fragments only. The nature of the fragments in the unit cell was probed by immuno-labelling with monovalent antibodies (Fab's), raised against the 75-kDa subunit from the Fe-S protein, followed by image analysis. We found at least four binding sites for the anti-(75-kDa subunit) Fab per unit cell, indicating the presence of at least four copies of the antigen. In order to account for these observations we postulate the hypothesis that the two-dimensional crystals obtained from complex I are composed of iron-sulphur protein molecules in an octameric arrangement.

Calcium-induced fusion of didodecylphosphate vesicles: the lamellar to hexagonal II (HII) phase transition.

Electron microscopic techniques have been employed to investigate the ability of didodecylphosphate vesicles (diameter approx. 900 A) to fuse in the presence of Ca2+. As revealed by negative staining, Ca2+ induces extensive fusion and large vesicles with diameters up to 7000 A are formed. In a process secondary to fusion, the fused vesicles display a tendency to flatten and are subsequently transformed into extended tubular structures. Freeze-fracture electron microscopy, in conjunction with 31P NMR and selected area electron diffraction measurements indicate that the tubes are packed in a hexagonal (HII) array and that the amphiphiles are converted from the lamellar to the hexagonal HII phase. The relationship between membrane fusion and the lamellar-to-hexagonal phase transition is discussed in terms of formation and abundance of transiently stable inverted micellar intermediates at contact regions between two interacting membranes. A model for the conversion of the (vesicular) lamellar into the (tubular) hexagonal HII phase is presented, taking into account the molecular shape of the amphiphile. The relevance of using simple synthetic amphiphiles as models for phospholipid bilayers and complex biomembrane behavior is briefly discussed.

Cyclic tetramolecular complexes of monoclonal antibodies: a new type of cross-linking reagent.

A simple and efficient procedure for the construction of bifunctional molecules is described and their use in a variety of applications documented. This procedure is based on our observation that mouse IgG1 monoclonal antibodies, when mixed with equimolar amounts of a high-affinity rat monoclonal antibody specific for mouse IgG1, yield uniform cyclic tetramolecular complexes each consisting of two mouse and two rat antibodies as shown by gel electrophoresis and electron microscopy. When solutions of two mouse antibodies (e.g. a and b) are mixed prior to the formation of complexes with the rat antibody, stable bispecific (a X b) complexes together with monospecific (a X a and b X b) complexes are obtained. Bispecific complexes prepared in this way were able to efficiently bind peroxidase to cell surface antigens, and to bind red blood cells to selected nucleated cell types present in heterogeneous populations. Tetrameric antibody complexes are more easily prepared than bispecific antibodies or bifunctional antibodies produced by transfection of myelomas with recombinant genes. They also have the advantage that the antigen-binding properties of the bivalent monoclonal antibodies are not compromised. Tetrameric antibody complexes thus represent a powerful new type of cross-linking reagent that may have a wide spectrum of applications in biology and medicine.

A model for the architecture of the hemocyanin from the arthropod Squilla mantis (Crustacea, Stomatopoda).

Squilla mantis hemocyanin is composed of two hexameric subunits but has electron microscopic profiles different from other bis-hexameric hemocyanins, e.g. Astacus and Homarus. We distinguished three different electron microscopic profiles of S. mantis hemocyanin: two sideviews and a topview. These profiles were studied using computer image alignment and correspondence analysis [Van Heel, M. and Frank, J. (1981) Ultramicroscopy 6, 187 - 194]. With the results of this analysis we were able to build a three-dimensional model for the quaternary structure of this hemocyanin. In this model the two hexamers are stacked in such a way that their hexagonal surfaces overlap to about 60% of their width. In the overlap area four subunits are arranged in two different interhexameric pairs, each forming a bridging area between the two hexamers.

Two-dimensional crystallization experiments.

Our experience in the growth of two-dimensional crystals of different proteins is presented. Polyethylene glycol was used to produce two-dimensional arrays of haemocyanin from O. vulgaris and of cholera toxin. The arrays showed a hexagonal close-packed structure of only randomly oriented molecules. The increase in protein concentration probably occurred too quickly to allow complete crystallization. Different two-dimensional arrays of hexameric haemocyanin molecules (from P. interruptus) were obtained by microdialysis through the specimen supporting film. A comparison was made with X-ray data. Two-dimensional tetrameric arrays of molecules, possibly rhodopsin, were seen in samples of bovine retinal rod outer segments in the presence of ammonium sulphate. Two-dimensional crystals of complex I (from bovine mitochondria) were prepared by dialysis in the presence of ammonium sulphate. A three-dimensional reconstruction was made from two tilt-series by computer filtration using the direct SIRT procedure. Finally, the possibility of computer crystallization using correlation techniques in combination with correspondence analysis is discussed.

Two-dimensional crystallization of bovine rhodopsin.

Bovine rhodopsin has been clustered into two-dimensional crystals in highly purified native rod disk membranes and studied with negative staining and transmission electron microscopy. The lattice is P2(1) with dimensions of 8.3 X 7.9 nm and interaxis angles of 86 +/- 3 degrees. 110 images of ordered areas were digitized and aligned with computer-correlation methods to calculate an average image with diffraction to the fourth order. The images were computer-filtered and reconstructed to approx. 2 nm resolution. When crystals appeared they covered 20-40% of the surface of the preparation and, since rhodopsin is at least 95% of the protein, there is no doubt that the crystals were due to rhodopsin. There appear to be two rhodopsin dimers per unit cell. Each rhodopsin molecules takes up about 7.5 nm2 of membrane area and is estimated to be associated with about 12 lipids on each side of the membrane. The membrane area found for bovine rhodopsin supports the rhodopsin origin of rarely seen but more highly ordered two-dimensional crystals found in detergent-treated frog rod membranes (Corless, J.M., McCaslin, D.R. and Scott, B.L. (1982) Proc. Natl. Acad. Sci. USA 79, 1116-1120). Furthermore, the rhodopsin membrane area is close to that of bacteriorhodopsin and is consistent with a seven transmembrane helix structure proposed for rhodopsin (for references see Dratz, E.A. and Hargrave, D.A. (1983) Trends Biochem. Sci. 8, 128-131). Crystallization was accomplished by lowering the pH to 5.5 near the isoelectric point of rhodopsin, raising the salt concentration of 2 M (NH4)2SO4, adding 5% glucose and 0.02% Hibitane (Ayerst), a cationic amphipathic antiseptic that favored crystal growth.

Electron microscopy of influenza virus. A comparison of negatively stained and ice-embedded particles.

An electron microscopical study was made of the influenza virus, type B/Hong Kong, in the unstained, frozen, hydrated state after quench-freezing in cooled liquid ethane. The results are compared with data from negatively stained specimens. It is shown that cryo-electron microscopy confirms and extends the data obtained by conventional methods. In particular, the virus is shown to be circular in projection with no indication of icosahedral symmetry, the lipid membrane is clearly resolved as a bi-layer and it is demonstrated that the distribution of material within the bi-layer is non-uniform, with a shell of more electron dense material surrounding a less dense central region. Neuraminidase spikes are not clearly distinguished from haemaglutinin spikes. The diameter of the complete B/Hong Kong virus was estimated from cryo-micrographs as 1270(+/- 70) A. Some preliminary data for influenza virus type A/X31 are presented.

Three-dimensional structure of bovine NADH:ubiquinone oxidoreductase of the mitochondrial respiratory chain.

We have studied the structure of bovine heart mitochondrial NADH:ubiquinone (Q) oxidoreductase (EC 1.6.99.3) by image analysis of electron micrographs. A three-dimensional reconstruction was calculated from a tilt-series of a two-dimensional crystal of the molecule. Our interpretation of the position of the molecule in the unit cell of the crystal is supported by additional (low-resolution) analysis of images of single molecules. The three-dimensional reconstruction was calculated with the aid of an iterative real-space reconstruction algorithm. The various projections used as input to the algorithm were obtained by averaging the images of the tilted crystal through a Fourier-space peak-filtering procedure. The reconstructed unit cell measures 15.2 X 15.2 nm in the plane of the two-dimensional crystal and has a height of 10-11 nm. The unit cell contains one molecule consisting of four large subunits. At the present resolution of about 1.3 nm in the untilted projection, these four monomers are seen as two dimers related by a two-fold axis. Two views of the single particles have been recognized; they are the top and side view of the building block of the crystal. After computer image alignment and correspondence analysis, clusters of similar particles have been averaged. In the averages an uneven stain distribution is seen around the molecules, which may result from preferential staining of hydrophilic parts of the molecule. The molecular mass of the whole molecule was determined from scanning transmission electron microscopy measurements as (1.6 +/- 0.2) X 10(6) daltons.