Ernst F.J. van Bruggen

Preparation of Two-Dimensional Crystals of Complex I and Image Analysis

Publisher Summary This chapter describes the preparation of the 2D crystals of complex-I and the image analysis. Several methods are available for the crystallization of membrane proteins. One approach to the problem of crystallizing membrane proteins is based on the classical methods of increasing the salt concentration. The chapter presents a discussion on characterization of the crystals. The crystals are built up from single tetrameric or pseudo-tetrameric protein molecules and are not formed by a rearrangement of molecules in vesicles or sheets, as is the case for some other membrane proteins. The removal of cholate during dialysis is essential for the formation of the crystals. The process of image processing includes (1) recording of the signal, (2) filtering of the 2D projections, and (3) 3D reconstruction. For very noisy images, a Fourier-filtered image should be used as a first reference instead of trying to align noisy fragments of the crystal directly on a noisy reference selected from the crystal.

Characterization of a low density cytoplasmic membrane subfraction isolated from Escherichia coli

We have used freeze fracture electron microscopy to study the distribution of membrane proteins in the cytoplasmic membrane of Escherichia coli W3110. While these proteins were distributed randomly at the growth temperature (37 degrees C), there was extensive protein lipid segregation when the temperature was lowered, resulting in bare patches containing no visible particles (protein), and areas of tightly packed or aggregated particles. To understand the segregation process, we have separated the bare patches from the particle rich membrane areas. Lysis of spheroplasts at 0 degrees C leads to cytoplasmic membrane fragments with different amounts of membrane particles per unit area; such fragments have been separated on isopycnic sucrose gradients. The bare patches occurred as low density membranes which were completely devoid of particles. They were compared to normal density cytoplasmic membranes with respect to fatty acid composition, protein distribution as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their content of several cytoplasmic membrane marker enzymes. The phospholipid to protein ratio of low density membranes was five times greater than that of normal membranes; unsaturated fatty acids were more abundant in the low density membranes. Most proteins had disappeared from the low density membranes. One protein, which had an apparent molecular weight of 26000 on sodium dodecyl sulfate gels appeared to be concentrated in the low density membranes; it accounted for about 50% of the total protein found in this membrane fraction. Of the cytoplasmic membrane markers tested, NADH oxidase and succinate dehydrogenase were excluded, while D-lactate dehydrogenase remained, and even appeared to be concentrated in the low density membranes. These results indicate that while most membrane proteins are associated with the fluid portion of the bilayer, some proteins evidently associate preferentially with phospholipids in the gel or frozen state.

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.

Structural characterization of the B800-850 and B875 light-harvesting antenna complexes from Rhodobacter sphaeroides by electron microscopy

Abstract The structure and aggregation behavior of B800–850 (LHII) and B875 (LHI) antenna complexes of Rhodobacter sphaeroides were studied by electron microscopy. Single molecular projections (top views and side views) of isolated particles were analyzed. The B800–850 complexes, isolated as 150 kDa particles, are cylindrical with a diameter of 8.5 nm and with a height of 6.5 nm. If corrected for attached detergent, the actual diameter in the plane of the membrane would be about 5.1 nm. Stain accumulation in the center of the structure indicates a small indentation, therefore a ring-shaped structure is expected. The size, shape and estimated mass give evidence for a B800–850 complex structure consisting of 4–6 αβ heterodimers, if in an αnβn ring-shaped configuration. The B875 complexes, isolated as 360 kDa particles, are dimeric units, but the two monomers are only loosely connected. A single B875 unit has a corrected diameter of about 5.2 nm and a height of 6.2 nm, similar to the B800–850 unit. Therefore, the B875 complex most likely has the same αnβn configuration. Image analysis of B800–850 projections points to a 3-or 6-fold symmetry in the top views, rather than a 4- or 5-fold symmetry. Therefore, our results are most compatible with an α6β6 configuration.

Localization of polypeptides in isolated chlorosomes from green phototrophic bacteria by immuno-gold labeling electron microscopy

Chlorosomes were prepared from Chloroflexus aurantiacus, Chlorobium limicola f. thiosulfatophilum and Prosthecochloris aestuarii by sucrose density gradient centrifugation. Two preparation methods were used. The first was applied to all three organisms and yielded chlorosomes without cytoplasmic membrane (CM) components. The second, which included a cross-linking step, was applied to Cf. aurantiacus and P. aestuarii only and yielded chlorosomes affixed to small inner CM patches. For the P. aestuarii sample this second method also yielded rod-elements, which were released from the inside of the chlorosomes. Antisera, raised against chlorosome polypeptide chains (18, 11 and 5.7 kDa), isolated from CM-free Cf. aurantiacus chlorosomes or a mixture of 24 and 24.5 kDa reaction center (RC) polypeptides isolated from Cf. aurantiacus inner CM, were incubated with the two different Cf. aurantiacus chlorosome samples and analyzed by gold-labeling electron microscopy (EM). Three antisera (anti-18 kDa, anti-11 kDa and anti-5.7 kDa) showed specific interaction with the chlorosomal envelope, whereas the anti-RC serum exhibited specificity for the CM. This result demonstrates for the first time that the 5.7 kDa polypeptide is in the chlorosomal envelope. Previous work had placed the polypeptide as a structural component of the intrachlorosomal rod-elements (Feick, R.G. and Fuller, R.C. (1984) Biochemistry 23, 3693–3700). Affinity-purified antibodies raised against a 7.5 kDa polypeptide isolated from Cb. limicola CM-free chlorosomes were incubated with Cb. limicola and P. aestuarii chlorosome samples. The antibodies showed considerable affinity for the chlorosomal envelopes but far less towards the CM components and hardly any for the rod-elements.

THE QUATERNARY STRUCTURE OF SEPIA-OFFICINALIS HEMOCYANIN

Abstract The haemocyanin (Hc) of Sepia officinalis is constituted of ten identical subunits ( M r about 390 000), which consist of eight functional (dioxygen-binding) units, designated by the letters a–h . A model is proposed for the quaternary structure of this HC, based on immuno-electron microscopy of whole molecules and on morphological studies of compact 1/5 molecules (consisting of two associated subunits). The immuno-electron microscopy was performed with IgG fractions containing antibodies specific for fragments abc, gh and de , respectively. Based on their general appearance, the immunocomplexes were divided into twelve types, whose occurernce was determined for the three IgG fractions. For each type of complex the location of the attachment sites of the IgGs was estimated in the cylindrical Hc molecule subdivided for this purpose into three regions (an upper, middle and lower third). The results indicate that functional units b to g form the wall of the cylindrical Hc molecule, while the two outer functional units of a and h are folded inside, thus forming a collar at the top and bottom of the cylinder. An experiment with a mixture of IgG fractions anti- abc and anti- gh showed that the ten subunits ( a–h ) lie parallel to each other, giving rise to an asymmetric molecule with, however, a symmetric appearance in the electron microscope.

The role of structurally diverse subunits in the assembly of three cheliceratan hemocyanins

Hemocyanins are high molecular mass respiratory copper proteins occuring in many a~hropods and molluscs [ 1 f. With the discovery of subunit heterogeneity in arthropod hemocyanins has come the question of how the presence of different subunits may influence the assembly and the functional properties of these proteins [2]. Certain subunits play specific roles in the assembly of the higher ordered structures [3-81. The generality of these studies was tested for Cheliceratan hemocyanins. The results obtained lead to the conclusion that there are structural correspondences between subunits of the hemocyanins from the horseshoe crab Limulus po~phem~ (eight-hexamer), the scorpion Androetonus australis (four-hexamer) and the tarantula Eurypelma californicum (four-hexamer). In all cases we can distinguish *hex~er-formers’ and ‘linkers’ needed to obtain the ‘multi-hexameric’ aggregates. It is even possible to form hybrid four-hexamers or a hybrid eight-hexamer from a mixture of subunits comb~g hex~er-foyers from one species with a linker from another species. Electron microscopy and thin-layer gel chromatography were used to monitor the assembly processes. 2. Materials and methods

Structure of NADH: Q Oxidoreductase from Bovine Heart Mitochondria Studied by Electron Microscopy

Two-dimensional crystalline arrays of NADH:Q oxidoreductase preparations have been obtained by microdiffusion of protein dissolved in detergent against a 15 mM sodium acetate buffer of pH 5.5 containing 10% (w/v) ammonium sulphate. Electron microscopy was used to study the structure of negatively stained crystals. Computer-reconstructed images were obtained by the Fourier peak filtering method. The crystals have p4 symmetry and a square unit cell with dimensions of 15.2 +/- 0.5 nm. The four asymmetric units in the unit cell form a single tetrameric molecule with a dimension in the third direction of 8.2 nm. It is concluded on the basis of the estimated molecular mass that each tetramer cannot contain more than only one FMN molecule. This implies that the tetramers possibly are only a part of Complex I, since there is much evidence that one functional enzyme molecule of Complex I contains two FMN molecules.

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.

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.