Jan F. L. Van Breemen

Solubilization of green plant thylakoid membranes with n-dodecyl, D-maltoside. Implications for the structural organization of the photosystem II, photosystem I, ATP synthase and cytochrome b6f complexes

A biochemical and structural analysis is presented of fractions that were obtained by a quick and mild solubilization of thylakoid membranes from spinach with the non-ionic detergent n-dodecyl-α,D-maltoside, followed by a partial purification using gel filtration chromatography. The largest fractions consisted of paired, appressed membrane fragments with an average diameter of about 360 nm and contain Photosystem II (PS II) and its associated light-harvesting antenna (LHC II), but virtually no Photosystem I, ATP synthase and cytochrome b6f complex. Some of the membranes show a semi-regular ordering of PS II in rows at an average distance of about 26.3 nm, and from a partially disrupted grana membrane fragment we show that the supercomplexes of PS II and LHC II represent the basic structural unit of PS II in the grana membranes. The numbers of free LHC II and PS II core complexes were very high and very low, respectively. The other macromolecular complexes of the thylakoid membrane occurred almost exclusively in dispersed forms. Photosystem I was observed in monomeric or multimeric PS I-200 complexes and there are no indications for free LHC I complexes. An extensive analysis by electron microscopy and image analysis of the CF0F1 ATP synthase complex suggests locations of the δ (on top of the F1 headpiece) and ∈ subunits (in the central stalk) and reveals that in a substantial part of the complexes the F1 headpiece is bended considerably from the central stalk. This kinking is very likely not an artefact of the isolation procedure and may represent the complex in its inactive, oxidized form.

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.

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.

Comparative electron microscopy and image analysis of oxy- and deoxy-hemocyanin from the spiny lobster Panulirus interruptus

Abstract Structural differences between oxy-hemocyanin and deoxy-hemocyanin from the spiny lobster P. interruptus were studied by electron microscopy and image analysis of negatively stained preparations. Projections of the hexameric P. interruptus hemocyanin from electron microscopy were compared with simulated projections of the high-resolution structure determined by X-ray crystallography previously. Image analysis was focused on the hexagonal views. Four independent data sets of hexagons (two of oxy- and two of deoxy-preparations) were analyzed and compared. Despite the small size of the projections, a resolution in the average images of better than 1.5 nm was obtained. A significant difference in stain modulation could be noticed between the oxy- and deoxy-preparations in two areas of the projections. The first difference in stain exclusion occurred in the center of the hexagonal views. The second is the shape of the domain 3 region. In the deoxy-images this region has a triangular shape, in the oxy-images it appears to be more rounded. The overall differences suggest a “breathing” of the hexamer upon oxygenation/deoxygenation.