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Electron microscopy of hemocyanins 1942–1986
Micron and Microscopica Acta, Vol. 17, No. 2, pp. 167—173, 1986. Printed in Great Britain.
0739-6260/86 $3.00+0.00 Pergamon Journals Ltd.
INVITED ARTICLE
ELECTRON MICROSCOPY OF HEMOCYANINS 1942-1986 E. F. J.
VAN BRUGGEN
Biochemisch Laboratorium, Rijksuniversiteit, Nijenborgh 16, 9747 AG Groningen, The Netherlands (Received 12 March 1986)
Abstract—Hemocyanins are chosen to demonstrate the contribution of electron microscopy to the study of protein molecules. The involvement of negative staining, computer-image processing and the first results with cryo-electron microscopy are discussed. Index key words: Electron microscopy, hemocyanin, negative staining, computer-image processing, cryoelectron microscopy.
INTRODUCTION ‘The results indicate that the electron microscope is very useful in the elucidation of the sizes and particularly the shapes of the larger protein molecules.’ This was written by Stanley and Anderson in 1942! We shall demonstrate, in this short review article, how true these words are for the study of hemocyanin architecture. Hemocyanins are respiratory proteins which occur freely dissolved in the hemolymph of certain species belonging to the phyla Arthropoda and Mollusca. Their molecular weights range from 450,000 to 14 million depending on the biological origin. They can dissociate into discrete, characteristic smaller units depending on, for example, the pH, ionic strength and the concentration ofdivalent cations. Within certain limits this dissociation is reversible. Hemocyanins from Arthropoda and Mollusca share many functional properties. However, their quaternary structure is very different. The size and structure of hemocyanin molecules make them very suitable for testing new methods of structural analysis. Eriksson-Quensel and Svedberg (1936) studied hemocyanins to demonstrate the possibility of using the analytical ultracentrifuge to determine the molecular weights of proteins. Early in 1960 we were looking for a suitable test object for electron microscopy other than a virus. Hemocyanins have been on our research 167
program ever since. This article describes the evolution of our structural knowledge of various hemocyanins in relation to the development of specimen preparation techniques, instrumental improvement and procedures for image processing.
THE GENERAL ARCHITECTURE OF HEMOCYANIN MOLECULES It is known from sedimentation analysis (Eriksson-Quensel and Svedberg, 1936), that hemocyanins of certain arthropodan origins occur in the hemolymph as molecules with measured sedimentation coefficients of about 16, 24, 34 and 60S. Nowadays we know that they are all built from subunits with a sedimentation coefficient of about 5S and a molecular weight ranging from 68,000 to 75,000. The 5S subunits represent single polypeptide chains, they bind two cuprous ions thus forming a center which can bind reversibly one molecule of oxygen. These 5S subunits are heterogeneous; five to possibly 12 different subunits have been found depending on the species. The smallest hemocyanin with a sedimentation coefficient of about 16S occurs in all spiny lobsters. It is a hexameric molecule with a molecular weight of 450,000. The larger arthro-
168
E. F. J. van Bruggen
podan hemocyanins occur as multiples of such hexameric molecules. Crabs and lobsters have two-, spiders and scorpions four- and the horseshoe crabs eight-hexameric hemocyanin molecules (van Bruggen et a!., 1981). The organization of molluscan hemocyanins is completely different. Depending on their biological origin they have sedimentation coefficients of about 50, 100 or 130S. Reversible dissociation is possible via 20S subunits into 11 S subunits (Van Holde and van Bruggen, 1971). The uS subunit comprises one single polypeptide chain arranged in eight different domains each with a molecular weight of 50,000—55,000 (Brouwer and Kuiper, 1973; Gielens et al., 1975). Each domain contains two cuprous ions which can bind in total one molecule of oxygen. The 50, 100 and 130S structures correspond to cylindrical molecules built from 10, 20 and 30 polypeptide chains, respectively. The diameter of the cylinders is about the same, their lengths are in the ratio of about 1:2:3. ELECTRON MICROSCOPY OF HEMOCYANINS Before 1960 Stanley and Anderson (1942) made the first electron micrographs of unstained hemocyanin molecules from the whelk Bus ycon canaliculatum,the horseshoe crab Limulus polyphemus and the snail Viviparus malleatus (Fig. 1). They analysed the shape and also measured the diameter of the molecules. Polson and Wyckoff (1947) photographed gold shadowed hemocyanin molecules of the whelk Busycon canaliculatum. They arrived at a model offour rod-shaped subunits with an aspect ratio of 3. Two or four of these rods are arranged in a parallel orientation. Schramm and Berger (1952) made electron micrographs of hemocyanin from the Roman snail Helix pomatia. The molecules were fixed in osmium tetroxide vapour and shadowed with palladium. Their conclusion was a brick-shaped molecule of 12.0 x 24.0 x 40.0 nm with a groove parallel to its longest side. The first dissociation step would occur along this groove into two identical half-molecules. Each half-molecule would then dissociate into four identical subunits~ (Fig. 2). Since 1960 Molluscan hemocyanins. The publication of
lig. I. Electron micrographs of unstained hemocyanin molecules reproduced from Stanley and Anderson (1942). (a) From the whelk Busycon canaliculatum, (b) from the horseshoe crab Limulus polyphemus.
-
-
L
-
-
b
Fig. 2. Molecular model of Roman snail (Helix pomatia) hemocyanin from Schramm and Berger (1952). (a) Whole molecule, (b) half molecule.
Brenner and Home (1959) describing a routine procedure for the negative staining of large virus molecules was of great Importance and equally applicable to the study of hemocyanins. When hemocyanin molecules of the Roman snail were studied in this way, they appeared as circles 35.0 nm in diameter or as rectangles
EM of Hemocyanins
169
35.0 x 38.0 nm (van Bruggen et a!., 1962). The circles showed five- or uo-fold rotational symmetry, while the rectangles were subdivided into six rows parallel to their shortest side (Fig. 3). They were clearly cylindrical molecules with a very specific substructure.
Fig. 4. Molecular model for hcmocyanin from the whelk Kelletia kelletia based on three-dimensional image reconstruction by Mellema and Klug (1972).
__________________
_________________________________
_____
_______
Each collar was made of five blobs of material. Further insight into the internal organization of these partly hollow cylindrical molecules was obtained by Siezen and van Bruggen (1974). A systematic study of dissociation products of hemocyanin from the Roman snail made it clear that the molecule first dissociates perpendicularly to the cylinder-axis into two identical halves. Each half molecule can be split along one ~-
________
_____
~ Fig. 3. Hemocyanin molecules from the Roman snail H. pomatta negatively stained with uranyl EDTA. The bar represents 50 nm.
The next problem was to analyse this substructure. Two methods were used: firstly the threedimensional image reconstruction procedure of Klug and De Rosier (1968) and secondly controlled dissociation of the molecules followed by electron microscopy of the dissociation products. The three-dimensional image reconstruction procedure for helical symmetry was applied to regularly arranged linear aggregates of cylindrical hemocyanin molecules from the whelk Kelletia kelletia (Mellema and Klug, 1972). The result was a hollow cylinder partly closed at both ends by a collar and possibly a cap (Fig. 4). The cylinder had point group symmetry 52. The wall of the cylinder consisted of 60 morphological units bounded by two sets of helical grooves,
to one polypeptide chain and can be observed as a necklace of eight globules at high pH and low ionic strength. An improved three-dimensional image reconstruction of the wall of the molecules was obtained with tubular helical polymers formed after mild proteolytic treatment of Roman snail f~-hemocyanin(van Breemen et a!., 1975). It was found that the two sets of helical grooves alternate in depth and that each morphological wall-unit of the Mellema and Kiug model consisted of two domains. Combination of all these data led to the schematic model shown in Fig. 5 for the arrangement of the 20 polypeptide chains within a cylindrical snail or whelk hemocyanin molecule (van Breemen et a!., 1979). The function of hemocyanin is the transport of oxygen. The native molecule of, for instance, Roman snail hemocyanin can be regarded as a large allosteric protein system containing 160 oxygen binding sites. The results of a biochemical
170
E. F. J. van Bruggen
1/2 1/
I
F’ _
Fig. 5. Schematic model for the arrangement of the polypep tide chains and for the way of dissociation of a cylindrical hemocyanin molecule.
study of this cooperative binding could be simply explained with a two-state model (van Driel and van Bruggen, 1975). Would it be possible to observe structural changes due to oxygenation by electron microscopy? Van Breemen et a!. (1979) carried out a statistical analysis of a large number of optical transforms taken from electron micrographs of tubular fJ-hemocyanin polymers in the oxygenated and deoxygenated states. Upon deoxygenation a significant decrease in diameter of the tubes was found with a concomitant increase in their length. The molecules were literally ‘breathing’! The observed changes were small and in the order of a few per cent. Art hropodan hemocyanins. Even more exciting is the story of the structural analysis of arthropodan hemocyanins. The most detailed knowledge has been obtained for the hexameric hemocyanin from the spiny lobster Panu!irus interruptus. Negative staining outlines these molecules as hexagons with a diameter of about 12.5 nm and as rectangles of about the same size with two sharp and two fuzzy edges (Fig. 6; Schepman, 1975). The molecular profiles show clear substructure leading to the proposal of several
Fig. 6. Hemocyanin molecules from the spiny lobster Panulirus interruptus negatively stained with uranyl acetate. The bar represents 50 nm. The insets show two views ofthe molecules at higher magnification.
molecular models. Fortunately it was possible to crystallize this hemocyanin and recently the structure was solved to 0.32 nm resolution using X-ray diffraction (Gaykema et al., 1984). The hexameric molecule is built from six subunits in a trigonal antiprismatic arrangement (Fig. 7). The projection of the molecule is a hexagon or a rectangle when reviewed parallel or perpendicular to the three-fold axis, respectively. The striking similarity between the X-ray data at 1.0 nm resolution and the data from electron microscopy is demonstrated in Fig. 8. Analysis of the rectangular profiles made it clear that the three-fold axis runs perpendicularly to the sharply contoured edges. This knowledge is important because it enables us to draw conclusions about the mutual orientation of the constituent hexamers in multi-hexameric hemocyanin molecules. Electron microscopy played an important role in both the determination of the various quaternary structures and the localiza-
EM of Hemocyanins
.
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b
171
view of their organization although with less detail has been presented by van Bruggen et al. (1981). Immuno-electron microscopy made it possible to localize the position of different subunits within these multi-hexameric molecules in cases of subunit heterogeneity. The first results were obtained by Lamy et a!. (1981) for the fourhexameric hemocyanin molecules from the scorpion Androctonus austra!is. Eight different types of subunit could be localized with specific monovalent Fab-fragments. As an example we show in Fig. 9 the localization of A. australis ‘subunit 6’.
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Fig. 7. Molecular model for hemocyanin from the spiny lobster P. interruptus based on X-ray diffraction data to 1.0 nm resolution (Gaykema et al., 1984). (a), (b) General views, (c) view parallel to the three-fold axis. Cd) view perpendicular to the three-fold axis.
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Fig. S. Molecular projection of P. inzerruprus hemocyanin parallel to the three-fold axis. (a) From X-ray diffraction data to 1.0 nm resolution, (b) from electron microscopy.
tion of the different types of monomeric and dimeric subunits within these structures, The application of advanced computer-image processing techniques, including correlation averaging and correspondence analysis, to single negatively stained molecules was of great value, Van Heel and Frank (1980) were the first to study the four-hexameric half-hemocyanin molecules from the horseshoe crab Limu!us po!yphemus in this way. They not only discovered that the molecules were lying in a face-up and a facedown orientation on the grid, but also that the hexamers on one diagonal axis of the slightly distorted rhombic structure were not in the same plane as the hexamers on the other diagonal axis. These computer-image processing procedures in combination with image simulations using X-ray data from spiny lobster hemocyanin will enable us to determine rather precisely the parameters (distances and angles) of the quaternary structure of such multi-hexameric hemocyanins. An over-
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I ig 9 Immuno ekL(ron micro’~.op~ applied to four htx.i menc hemocyanin molecules from the scorpion Androctonus astralis. Localization of ‘subunit 6’ with specific antisubunit 6 Fab-fragments. The bar represents 50 nm
In the meantime protein chemists collected amino acid sequence data for seven different subunits from arthropodan hemocyanins (Linzen eta!., 1985). Comparison of the data showed many conserved residues and extensive regions of near identity. The correspondence could be matched closely with the three-dimensional structure shown by X-ray crystallography for spiny lobster hemocyanin. The polypeptide architecture appeared to be the same for all arthropodan hemocyanins. Perhaps it will now be possible to study the hexamer interactions in multi-hexameric hemocyanins in more detail by combining the X-ray and EM data with the amino acid sequence. CRYOELECTRON MICROSCOPY OF HEMOCYANINS All the electron microscopical results presented so far have been obtained using the
172
E. F. J. van Bruggen
technique ofnegative staining or shadowing with staining we now observe not only two, but many a maximum resolution of 2.0 nm. Either visually different projections of the cylindrical molecules. or in combination with computer-image process- The dimensions of the cylinders are ca ing techniques, structural data have been col- 29.0 x 38.0 nm compared with 35.0 x 38.0 nm lected which could be correlated very well with after negative staining. The difference probably information from other structural or biochemical results from flattening during negative staining. techniques. It would still be of great scientific Some substructure is visible although with low value to be able to study these molecules close to contrast. their native state at a resolution of say 0.5 nm. We wonder whether it will be possible to detect Cryo-electron microscopy seems to fulfil these differences between oxy- and deoxyhemocyanin demands. The procedure of Adrian et a!. (1984) molecules using cryo-electron microscopy. We used to study unfixed unstained biomacromole- have startedexperiments with periodic hemocyacules in a frozen, hydrated state seems very nm structures, for example, H. pomatia fl-hemopromising. cyanin tubes and thin crystals of P. interruptus Using similar techniques we have recently hemocyanin hexamers. In the last case frozen obtained cryo-electron micrographs of the cylin- hydrated crystals of oxyhemocyanin give elecdrical hemocyanin molecules from the whelk K. tron diffraction patterns extending to 0.29 nm. kel!etia, of tubular polymers from the snail H. The resolution of corresponding images was pomatia and the eight-hexameric molecules of the 1.5—2.0 nm, probably due to vibration of the horseshoe crab L. po!yphemus (Schutter et a!., specimen cooling holder. 1986). As an example we present here a cryoelectron micrograph of K. ke!!etia hemocyanin molecules (Fig. 10). Unlike the case with negative CLOSING REMARKS Here ends this hemocyanin story for the moment! We hope that we have made it clear that electron microscopy in all its variations has played an essential role in these studies and certainly will do so in the years to come. Hemocyanin was taken as an example, similar stories can be written for many other biomacromolecular assemblies. Acknowlegements—We thank all our many colleagues who collaborated within this hemocyanin program; Dr. T. Wichertjes for her help with the illustrations and K. Gilissen for printing and mounting the photographs. These studies are supported by the Netherlands Foundation for Chemical Research (SON) with financial aid from the Netherlands Organization for the Advancement ofPure Research (ZWO).
REFERENCES
Fig. 0. Cryo-electron micrograph of frozen-hydrated. unstained,unfixed hemocyanin molecules from the whelk A. kelletia.
Adrian, M., Dubochet, J., Lepault, J. and McDowell, A. W., 1984. Cryo-electron microscopy of viruses. Nature, Lond., 308: 32—36. Breemen, J. F. L. van, Wichertjes, T., Muller, M. F. J., Driel, R. van and Bruggen, E. F. J. van, 1975. Tubular polymers derived from Helix pomatia fl-hemocyanin. Eur. J. Biochem., 60: 129—135. Breemen, J. F. L. van, Ploegman, J. H. and Bruggen, E. F. J. van, 1979. Structure of Helix pomatia oxy-~l-hemocyanin and deoxy-/J-hemocyanin tubular polymers. Eur. J. Biochem., 100: 61—65. Brenner, S. and Home, R. W., 1959. A negative staining method for high resolution electron microscopy ofviruses. Biochem. biophys. Acta, 34: 103—110.
EM of Hemocyanins Brouwer, M. and Kuiper, H. A., 1973. Molecular weight analysis of Helix pomatia hemocyanin in guanidinehydrochloride, urea and sodium dodecylsulfate. Eur. J. Biochem., 35: 428—435. Bruggen, E. F. J. van, Wiebenga, E. H. and Gruber, M., 1962. Structure and properties of hemocyanins: I. Electron micrographs of hemocyanin and apohemocyanin from Helix pomatia at different pH values. J. molec. Biol., 4: 1—7. Bruggen, F. F. J. van, Schutter, W. G., Breemen, J. F. L. van, Bijlholt, M. M. C. and Wichertjes, T., 1981. Arthropodan and molluscan hemocyanins. In: Electron Microscopy of Proteins, Harris, J. R. (ed.), Academic Press, London, Vol. 1, 1—38. Driel, R. van and Bruggen, E. F. J. van, 1975. Functional properties ofchemically modified hemocyanin. Fixation of hemocyanin in the low and the high oxygen affinity state by reaction with a bifunctional ester. Biochemistry, 14: 730—735. Eriksson-Quensel, 1.-B. and Svedberg, T., 1936. The moleculam weights and pH-stability regions of the hemocyanins. Biol. Bull., 71: 498—548. Gaykema, W. P. J., Hol, W. G. J., Vereijken, J. M., Soeter, N. M., Bak, H. J. and Beintema, J. J., 1984. 3.2 A structure of the copper-binding, oxygen-carrying protein Panulirus interruplus haemocyanin. Nature, Lond., 309: 23—29. Gielens, C., Preaux, G. and Lontie, R., 1975. Limited trypsinolysis of fl-hemocyanin of Helix pomatia. Eur. J. Biochem., 60: 271—280. Heel, M. van and Frank, J., 1981. Use of multivariate statistics in analysing the images of biological macromolecules. Ultramicroscopy, 6: 187—194. Lamy, J., Bijlholt, M. M. C., Sizaret, P.-Y., Lamy, J. and Bruggen, E. F. J. van, 1981. Quaternary structure of
173
scorpion (Androctonus austrails) hemocyanin. Localization of subunits with immunological methods and electron microscopy. Biochemistry, 20: 1849—1856. Linzen, B., Soeter, N. M., Riggs, A. F., Schneider, H-i., Schartau, W., Moore, M. D., Yokota, E., Behrens, P. Q., Nakashima, H., Takagi, T., Nemoto, T., Vereijken, J. M., Bak, H. J.,Beintema, J. J., Volbeda, A., Gaykema, W. P. J. and Hol, W. G. J., 1985. The structure of Arthropod hemocyanins. Science, 229: 5 19—524. Mellema, J. E. and KIug, A., 1972. Quatemnary structure of Gastropod haemocyanin. Nature, Lond., 239: 146—150. PoIson, A. and Wyckoff, R. W. G., 1947. Shape of haemocyanin molecules. Nature, Lond., 160: 153—154. Schepman, A. M. H., 1975. X-Ray diffraction and electron microscopy of Panulirus interruptus hemocyanin. Doctors thesis, Groningen, 57—75. Schramm, G. and Berger, G., 1952. Elektronenmikroskopische Untersuchungen über die Struktur des Hämocyanins von Helix pomatia. Z. Naturforsch., 7b: 286—288. Schutter, W. G., Keegstra, W., Booy, F., Haker, J. and Bruggen, E. F. J. van, 1986. STEM and cryo-TEM of Limulus and Kelletia hemocyanin. In: Invertebrate Oxygen Carriers, in press. Siezen, R. J. and Bruggen, E. F. J. van, 1974. Structure and properties of hemocyanins: XII. Electron microscopy of dissociation products of Helix pomatia a-hemocyanin: quatemnary structure. J. molec. Biol., 90: 77—89. Stanley, W. and Anderson, T., 1942. Electron micrographs of protein molecules. J. biol. Chem., 146: 25—33. Van Holde, K. E. and Bruggen, E. F. J. van, 1971. The hemocyanins. In: Biological Macromolecules Series, Timasheff, S. N. and Fasman, G. D. (eds.), Marcel Dekker, New York, Vol. 5, part A, 1—53.
0739-6260/86 $3.00+0.00 Pergamon Journals Ltd.
INVITED ARTICLE
ELECTRON MICROSCOPY OF HEMOCYANINS 1942-1986 E. F. J.
VAN BRUGGEN
Biochemisch Laboratorium, Rijksuniversiteit, Nijenborgh 16, 9747 AG Groningen, The Netherlands (Received 12 March 1986)
Abstract—Hemocyanins are chosen to demonstrate the contribution of electron microscopy to the study of protein molecules. The involvement of negative staining, computer-image processing and the first results with cryo-electron microscopy are discussed. Index key words: Electron microscopy, hemocyanin, negative staining, computer-image processing, cryoelectron microscopy.
INTRODUCTION ‘The results indicate that the electron microscope is very useful in the elucidation of the sizes and particularly the shapes of the larger protein molecules.’ This was written by Stanley and Anderson in 1942! We shall demonstrate, in this short review article, how true these words are for the study of hemocyanin architecture. Hemocyanins are respiratory proteins which occur freely dissolved in the hemolymph of certain species belonging to the phyla Arthropoda and Mollusca. Their molecular weights range from 450,000 to 14 million depending on the biological origin. They can dissociate into discrete, characteristic smaller units depending on, for example, the pH, ionic strength and the concentration ofdivalent cations. Within certain limits this dissociation is reversible. Hemocyanins from Arthropoda and Mollusca share many functional properties. However, their quaternary structure is very different. The size and structure of hemocyanin molecules make them very suitable for testing new methods of structural analysis. Eriksson-Quensel and Svedberg (1936) studied hemocyanins to demonstrate the possibility of using the analytical ultracentrifuge to determine the molecular weights of proteins. Early in 1960 we were looking for a suitable test object for electron microscopy other than a virus. Hemocyanins have been on our research 167
program ever since. This article describes the evolution of our structural knowledge of various hemocyanins in relation to the development of specimen preparation techniques, instrumental improvement and procedures for image processing.
THE GENERAL ARCHITECTURE OF HEMOCYANIN MOLECULES It is known from sedimentation analysis (Eriksson-Quensel and Svedberg, 1936), that hemocyanins of certain arthropodan origins occur in the hemolymph as molecules with measured sedimentation coefficients of about 16, 24, 34 and 60S. Nowadays we know that they are all built from subunits with a sedimentation coefficient of about 5S and a molecular weight ranging from 68,000 to 75,000. The 5S subunits represent single polypeptide chains, they bind two cuprous ions thus forming a center which can bind reversibly one molecule of oxygen. These 5S subunits are heterogeneous; five to possibly 12 different subunits have been found depending on the species. The smallest hemocyanin with a sedimentation coefficient of about 16S occurs in all spiny lobsters. It is a hexameric molecule with a molecular weight of 450,000. The larger arthro-
168
E. F. J. van Bruggen
podan hemocyanins occur as multiples of such hexameric molecules. Crabs and lobsters have two-, spiders and scorpions four- and the horseshoe crabs eight-hexameric hemocyanin molecules (van Bruggen et a!., 1981). The organization of molluscan hemocyanins is completely different. Depending on their biological origin they have sedimentation coefficients of about 50, 100 or 130S. Reversible dissociation is possible via 20S subunits into 11 S subunits (Van Holde and van Bruggen, 1971). The uS subunit comprises one single polypeptide chain arranged in eight different domains each with a molecular weight of 50,000—55,000 (Brouwer and Kuiper, 1973; Gielens et al., 1975). Each domain contains two cuprous ions which can bind in total one molecule of oxygen. The 50, 100 and 130S structures correspond to cylindrical molecules built from 10, 20 and 30 polypeptide chains, respectively. The diameter of the cylinders is about the same, their lengths are in the ratio of about 1:2:3. ELECTRON MICROSCOPY OF HEMOCYANINS Before 1960 Stanley and Anderson (1942) made the first electron micrographs of unstained hemocyanin molecules from the whelk Bus ycon canaliculatum,the horseshoe crab Limulus polyphemus and the snail Viviparus malleatus (Fig. 1). They analysed the shape and also measured the diameter of the molecules. Polson and Wyckoff (1947) photographed gold shadowed hemocyanin molecules of the whelk Busycon canaliculatum. They arrived at a model offour rod-shaped subunits with an aspect ratio of 3. Two or four of these rods are arranged in a parallel orientation. Schramm and Berger (1952) made electron micrographs of hemocyanin from the Roman snail Helix pomatia. The molecules were fixed in osmium tetroxide vapour and shadowed with palladium. Their conclusion was a brick-shaped molecule of 12.0 x 24.0 x 40.0 nm with a groove parallel to its longest side. The first dissociation step would occur along this groove into two identical half-molecules. Each half-molecule would then dissociate into four identical subunits~ (Fig. 2). Since 1960 Molluscan hemocyanins. The publication of
lig. I. Electron micrographs of unstained hemocyanin molecules reproduced from Stanley and Anderson (1942). (a) From the whelk Busycon canaliculatum, (b) from the horseshoe crab Limulus polyphemus.
-
-
L
-
-
b
Fig. 2. Molecular model of Roman snail (Helix pomatia) hemocyanin from Schramm and Berger (1952). (a) Whole molecule, (b) half molecule.
Brenner and Home (1959) describing a routine procedure for the negative staining of large virus molecules was of great Importance and equally applicable to the study of hemocyanins. When hemocyanin molecules of the Roman snail were studied in this way, they appeared as circles 35.0 nm in diameter or as rectangles
EM of Hemocyanins
169
35.0 x 38.0 nm (van Bruggen et a!., 1962). The circles showed five- or uo-fold rotational symmetry, while the rectangles were subdivided into six rows parallel to their shortest side (Fig. 3). They were clearly cylindrical molecules with a very specific substructure.
Fig. 4. Molecular model for hcmocyanin from the whelk Kelletia kelletia based on three-dimensional image reconstruction by Mellema and Klug (1972).
__________________
_________________________________
_____
_______
Each collar was made of five blobs of material. Further insight into the internal organization of these partly hollow cylindrical molecules was obtained by Siezen and van Bruggen (1974). A systematic study of dissociation products of hemocyanin from the Roman snail made it clear that the molecule first dissociates perpendicularly to the cylinder-axis into two identical halves. Each half molecule can be split along one ~-
________
_____
~ Fig. 3. Hemocyanin molecules from the Roman snail H. pomatta negatively stained with uranyl EDTA. The bar represents 50 nm.
The next problem was to analyse this substructure. Two methods were used: firstly the threedimensional image reconstruction procedure of Klug and De Rosier (1968) and secondly controlled dissociation of the molecules followed by electron microscopy of the dissociation products. The three-dimensional image reconstruction procedure for helical symmetry was applied to regularly arranged linear aggregates of cylindrical hemocyanin molecules from the whelk Kelletia kelletia (Mellema and Klug, 1972). The result was a hollow cylinder partly closed at both ends by a collar and possibly a cap (Fig. 4). The cylinder had point group symmetry 52. The wall of the cylinder consisted of 60 morphological units bounded by two sets of helical grooves,
to one polypeptide chain and can be observed as a necklace of eight globules at high pH and low ionic strength. An improved three-dimensional image reconstruction of the wall of the molecules was obtained with tubular helical polymers formed after mild proteolytic treatment of Roman snail f~-hemocyanin(van Breemen et a!., 1975). It was found that the two sets of helical grooves alternate in depth and that each morphological wall-unit of the Mellema and Kiug model consisted of two domains. Combination of all these data led to the schematic model shown in Fig. 5 for the arrangement of the 20 polypeptide chains within a cylindrical snail or whelk hemocyanin molecule (van Breemen et a!., 1979). The function of hemocyanin is the transport of oxygen. The native molecule of, for instance, Roman snail hemocyanin can be regarded as a large allosteric protein system containing 160 oxygen binding sites. The results of a biochemical
170
E. F. J. van Bruggen
1/2 1/
I
F’ _
Fig. 5. Schematic model for the arrangement of the polypep tide chains and for the way of dissociation of a cylindrical hemocyanin molecule.
study of this cooperative binding could be simply explained with a two-state model (van Driel and van Bruggen, 1975). Would it be possible to observe structural changes due to oxygenation by electron microscopy? Van Breemen et a!. (1979) carried out a statistical analysis of a large number of optical transforms taken from electron micrographs of tubular fJ-hemocyanin polymers in the oxygenated and deoxygenated states. Upon deoxygenation a significant decrease in diameter of the tubes was found with a concomitant increase in their length. The molecules were literally ‘breathing’! The observed changes were small and in the order of a few per cent. Art hropodan hemocyanins. Even more exciting is the story of the structural analysis of arthropodan hemocyanins. The most detailed knowledge has been obtained for the hexameric hemocyanin from the spiny lobster Panu!irus interruptus. Negative staining outlines these molecules as hexagons with a diameter of about 12.5 nm and as rectangles of about the same size with two sharp and two fuzzy edges (Fig. 6; Schepman, 1975). The molecular profiles show clear substructure leading to the proposal of several
Fig. 6. Hemocyanin molecules from the spiny lobster Panulirus interruptus negatively stained with uranyl acetate. The bar represents 50 nm. The insets show two views ofthe molecules at higher magnification.
molecular models. Fortunately it was possible to crystallize this hemocyanin and recently the structure was solved to 0.32 nm resolution using X-ray diffraction (Gaykema et al., 1984). The hexameric molecule is built from six subunits in a trigonal antiprismatic arrangement (Fig. 7). The projection of the molecule is a hexagon or a rectangle when reviewed parallel or perpendicular to the three-fold axis, respectively. The striking similarity between the X-ray data at 1.0 nm resolution and the data from electron microscopy is demonstrated in Fig. 8. Analysis of the rectangular profiles made it clear that the three-fold axis runs perpendicularly to the sharply contoured edges. This knowledge is important because it enables us to draw conclusions about the mutual orientation of the constituent hexamers in multi-hexameric hemocyanin molecules. Electron microscopy played an important role in both the determination of the various quaternary structures and the localiza-
EM of Hemocyanins
.
4
-‘
a
b
171
view of their organization although with less detail has been presented by van Bruggen et al. (1981). Immuno-electron microscopy made it possible to localize the position of different subunits within these multi-hexameric molecules in cases of subunit heterogeneity. The first results were obtained by Lamy et a!. (1981) for the fourhexameric hemocyanin molecules from the scorpion Androctonus austra!is. Eight different types of subunit could be localized with specific monovalent Fab-fragments. As an example we show in Fig. 9 the localization of A. australis ‘subunit 6’.
,~
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Fig. 7. Molecular model for hemocyanin from the spiny lobster P. interruptus based on X-ray diffraction data to 1.0 nm resolution (Gaykema et al., 1984). (a), (b) General views, (c) view parallel to the three-fold axis. Cd) view perpendicular to the three-fold axis.
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Fig. S. Molecular projection of P. inzerruprus hemocyanin parallel to the three-fold axis. (a) From X-ray diffraction data to 1.0 nm resolution, (b) from electron microscopy.
tion of the different types of monomeric and dimeric subunits within these structures, The application of advanced computer-image processing techniques, including correlation averaging and correspondence analysis, to single negatively stained molecules was of great value, Van Heel and Frank (1980) were the first to study the four-hexameric half-hemocyanin molecules from the horseshoe crab Limu!us po!yphemus in this way. They not only discovered that the molecules were lying in a face-up and a facedown orientation on the grid, but also that the hexamers on one diagonal axis of the slightly distorted rhombic structure were not in the same plane as the hexamers on the other diagonal axis. These computer-image processing procedures in combination with image simulations using X-ray data from spiny lobster hemocyanin will enable us to determine rather precisely the parameters (distances and angles) of the quaternary structure of such multi-hexameric hemocyanins. An over-
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I ig 9 Immuno ekL(ron micro’~.op~ applied to four htx.i menc hemocyanin molecules from the scorpion Androctonus astralis. Localization of ‘subunit 6’ with specific antisubunit 6 Fab-fragments. The bar represents 50 nm
In the meantime protein chemists collected amino acid sequence data for seven different subunits from arthropodan hemocyanins (Linzen eta!., 1985). Comparison of the data showed many conserved residues and extensive regions of near identity. The correspondence could be matched closely with the three-dimensional structure shown by X-ray crystallography for spiny lobster hemocyanin. The polypeptide architecture appeared to be the same for all arthropodan hemocyanins. Perhaps it will now be possible to study the hexamer interactions in multi-hexameric hemocyanins in more detail by combining the X-ray and EM data with the amino acid sequence. CRYOELECTRON MICROSCOPY OF HEMOCYANINS All the electron microscopical results presented so far have been obtained using the
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E. F. J. van Bruggen
technique ofnegative staining or shadowing with staining we now observe not only two, but many a maximum resolution of 2.0 nm. Either visually different projections of the cylindrical molecules. or in combination with computer-image process- The dimensions of the cylinders are ca ing techniques, structural data have been col- 29.0 x 38.0 nm compared with 35.0 x 38.0 nm lected which could be correlated very well with after negative staining. The difference probably information from other structural or biochemical results from flattening during negative staining. techniques. It would still be of great scientific Some substructure is visible although with low value to be able to study these molecules close to contrast. their native state at a resolution of say 0.5 nm. We wonder whether it will be possible to detect Cryo-electron microscopy seems to fulfil these differences between oxy- and deoxyhemocyanin demands. The procedure of Adrian et a!. (1984) molecules using cryo-electron microscopy. We used to study unfixed unstained biomacromole- have startedexperiments with periodic hemocyacules in a frozen, hydrated state seems very nm structures, for example, H. pomatia fl-hemopromising. cyanin tubes and thin crystals of P. interruptus Using similar techniques we have recently hemocyanin hexamers. In the last case frozen obtained cryo-electron micrographs of the cylin- hydrated crystals of oxyhemocyanin give elecdrical hemocyanin molecules from the whelk K. tron diffraction patterns extending to 0.29 nm. kel!etia, of tubular polymers from the snail H. The resolution of corresponding images was pomatia and the eight-hexameric molecules of the 1.5—2.0 nm, probably due to vibration of the horseshoe crab L. po!yphemus (Schutter et a!., specimen cooling holder. 1986). As an example we present here a cryoelectron micrograph of K. ke!!etia hemocyanin molecules (Fig. 10). Unlike the case with negative CLOSING REMARKS Here ends this hemocyanin story for the moment! We hope that we have made it clear that electron microscopy in all its variations has played an essential role in these studies and certainly will do so in the years to come. Hemocyanin was taken as an example, similar stories can be written for many other biomacromolecular assemblies. Acknowlegements—We thank all our many colleagues who collaborated within this hemocyanin program; Dr. T. Wichertjes for her help with the illustrations and K. Gilissen for printing and mounting the photographs. These studies are supported by the Netherlands Foundation for Chemical Research (SON) with financial aid from the Netherlands Organization for the Advancement ofPure Research (ZWO).
REFERENCES
Fig. 0. Cryo-electron micrograph of frozen-hydrated. unstained,unfixed hemocyanin molecules from the whelk A. kelletia.
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