From protons to OXPHOS supercomplexes and Alzheimer's disease: structure-dynamics-function relationships of energy-transducing membranes.

By the elucidation of high-resolution structures the view of the bioenergetic processes has become more precise. But in the face of these fundamental advances, many problems are still unresolved. We have examined a variety of aspects of energy-transducing membranes from large protein complexes down to the level of protons and functional relevant picosecond protein dynamics. Based on the central role of the ATP synthase for supplying the biological fuel ATP, one main emphasis was put on this protein complex from both chloroplast and mitochondria. In particular the stoichiometry of protons required for the synthesis of one ATP molecule and the supramolecular organisation of ATP synthases were examined. Since formation of supercomplexes also concerns other complexes of the respiratory chain, our work was directed to unravel this kind of organisation, e.g. of the OXPHOS supercomplex I(1)III(2)IV(1), in terms of structure and function. Not only the large protein complexes or supercomplexes work as key players for biological energy conversion, but also small components as quinones which facilitate the transfer of electrons and protons. Therefore, their location in the membrane profile was determined by neutron diffraction. Physico-chemical features of the path of protons from the generators of the electrochemical gradient to the ATP synthase, as well as of their interaction with the membrane surface, could be elucidated by time-resolved absorption spectroscopy in combination with optical pH indicators. Diseases such as Alzheimer's dementia (AD) are triggered by perturbation of membranes and bioenergetics as demonstrated by our neutron scattering studies.

Projection structure of the monomeric porin OmpG at 6 A resolution.

The Escherichia coli porin OmpG, which acts as an efficient unspecific channel for mono-, di- and trisaccharides, has been purified and crystallized in two dimensions. Projection maps of two different crystal forms of OmpG at 6 A resolution show that the protein has a beta-barrel structure characteristic for outer membrane proteins, and that it does not form trimers, unlike most other porins such as OmpF and OmpC, but appears in monomeric form. The size of the barrel is approximately 2.5 nm, indicating that OmpG may consist of 14 beta-strands. The projection map suggests that the channel is restricted by internal loops.

Parameters affecting specimen flatness of two-dimensional crystals for electron crystallography.

The flatness of two-dimensional (2D) crystals on the support film is a critical factor in protein electron crystallography. The influence of the carbon support film and of different grid makes and materials on flatness was investigated, using as a criterion the sharpness of diffraction spots perpendicular to the tilt axis of electron diffraction patterns of purple membrane tilted in the microscope at 45 degrees. In a quantitative test, carbon film that had been evaporated without sparks forming gave a much larger proportion of flat crystals than "sparked" carbon. Titanium grids were superior to copper, probably because they introduce less cryo-crinkling of the carbon film when the sample is cooled to liquid nitrogen temperature, as their thermal expansion coefficient is closer to that of carbon. While the molybdenum grids from Plano were unsuitable for data collection because of their tendency of break the carbon, molybdenum grids from Pacific GridTech gave a much larger yield of flat crystals than the titanium grids. Scanning electron microscope images of the grids as supplied by the manufacturer showed that the Plano grids had very narrow and irregular grid bars, while the Pacific GridTech grids were very smooth with a large surface-to-hole ratio.

Structure of the bacteriorhodopsin mutant F219L N intermediate revealed by electron crystallography.

Bacteriorhodopsin is a light-driven proton pump in halobacteria that forms crystalline patches in the cell membrane. Isomerization of the bound retinal initiates a photocycle resulting in the extrusion of a proton. An electron crystallographic analysis of the N intermediate from the mutant F219L gives a three-dimensional view of the large conformational change that occurs on the cytoplasmic side after deprotonation of the retinal Schiff base. Helix F, together with helix E, tilts away from the center of the molecule, causing a shift of approximately 3 A at the EF loop. The top of helix G moves slightly toward the ground state location of helix F. These movements open a water-accessible channel in the protein, enabling the transfer of a proton from an aspartate residue to the Schiff base. The movement of helix F toward neighbors in the crystal lattice is so large that it would not allow all molecules to change conformation simultaneously, limiting the occupancy of this state in the membrane to 33%. This explains photocooperative phenomena in the purple membrane.

A three-dimensional difference map of the N intermediate in the bacteriorhodopsin photocycle: part of the F helix tilts in the M to N transition.

The N intermediate of the bacteriorhodopsin photocycle was trapped for electron diffraction studies in glucose-embedded specimens of the site-directed mutant Phe219 --> Leu. At neutral pH, the N-bR difference Fourier transform infrared spectrum of this mutant is indistinguishable from published difference spectra obtained for wild-type bacteriorhodopsin at alkaline pH. An electron diffraction difference map of the N intermediate in projection shows large differences near the F and the G helix, which are very similar to the features seen in the M intermediates of the Asp96 --> Gly mutant [Subramaniam et al. (1993) EMBO J. 12, 1-8]. This similarity was anticipated on the basis of Fourier transform infrared data, which have shown that the M intermediate trapped in Asp96 mutants already has the protein structure of the N intermediate [Sasaki et al. (1992) J. Biol. Chem. 267, 20782-20786]. A preliminary three-dimensional difference map of the N intermediate, calculated from electron diffraction data of samples tilted at 25 degrees, clearly shows that the change on the F helix consists of an outward movement of the cytoplasmic end of the helix. In addition, the cytoplasmic side of the G helix moves or becomes more ordered. Comparison with published difference maps of the M intermediate indicates that the F helix tilt occurs in the M to N transition, but the G helix change represents an earlier step in the photocycle.

Two progressive substrates of the M-intermediate can be identified in glucose-embedded, wild-type bacteriorhodopsin.

Glucose-embedded bacteriorhodopsin shows M-intermediates with different Amide I infrared bands when samples are illuminated at 240 or 260 K, in contrast with fully hydrated samples where a single M-intermediate is formed at all temperatures. In hydrated, but not in glucose-embedded specimens, the N intermediate is formed together with M at 260 K. Both Fourier transform infrared and electron diffraction data from glucose-embedded bacteriorhodopsin suggest that at 260 K a mixture is formed of the M-state that is trapped at 240 K, and a different M-intermediate (MN) that is also formed by mutant forms of bacteriorhodopsin that lack a carboxyl group at the 96 position, necessary for the M to N transition. The fact that an MN species is trapped in glucose-embedded, wild-type bacteriorhodopsin suggests that the glucose samples lack functionally important water molecules that are needed for the proton transfer aspartate 96 to the Schiff base (and, thus, to form the N-intermediate); thus, aspartate 96 is rendered ineffective as a proton donor.

The bacteriorhodopsin photocycle: direct structural study of two substrates of the M-intermediate.

Changes in protein structure that occur during the formation of the M photointermediate of bacteriorhodopsin can be directly visualized by electron diffraction techniques. A modified preparation technique for glucose-embedded crystals was employed to ensure sufficient hydration of the crystals, which was needed for the formation of the M intermediate at low temperature. Samples containing a high percentage of the M intermediate were trapped by rapidly cooling the crystals with liquid nitrogen after illumination with filtered green light at 240 and 260 K, respectively. Difference Fourier projection maps are presented for the M intermediates formed at these two temperatures. The diffraction data clearly show that statistically significant structural changes occur upon formation of the M intermediate at 240 K and then further upon formation of the second specimen that is produced at 260 K.

Specimen flatness of glucose-embedded biological materials for electron crystallography is affected significantly by the choice of carbon evaporation stock.

Imperfect specimen flatness can be a significant limitation in the application of electron crystallography to high-resolution structure analysis of biological macromolecules. We now report that the choice of solid carbon stock that is used to make evaporated carbon films can have a very great effect on the preparation of flat specimens of glucose-embedded purple membrane. The degree of purity of the carbon does not seem to be the controlling factor, and other likely factors such as the type of mica used as a substrate, the evaporation apparatus used (and its limiting vacuum), and the use of a continuous versus an interrupted evaporation protocol do not have a discernible influence. The physical or chemical basis for the observed differences in specimen flatness is still unknown; however, the important conclusion that we can communicate at this point is that the choice of evaporating material does have a major effect on the flatness of purple membrane, the specimen used here. The implication is that different sources of carbon stock should be tried whenever difficulty is encountered in the preparation of suitably flat specimens of biological macromolecules.

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.

In vivo crystal formation in Escherichia coli of an over-expressed soluble form of penicillin-binding protein 5.

Accumulation of either native membrane-bound or soluble variants of PBP5 over-expressed in the cytoplasm was investigated by electron microscopy of ultra-thin sections. One of the soluble forms of PBP5 (PBP5s353) formed well-ordered crystals inside the cells. Cells sectioned perpendicular to their long axis showed a diamond-shaped crystal whereas cells cut parallel to their long axis contained a long, narrow crystal. In both sectioning directions an ordered ultrastructure was visible as shown by optical diffraction. Computer processing was used to enhance the crystal images. From this the unit cell parameters were calculated as a = 7.6 nm, b = 4 nm, c = 4.2 nm, gamma = 75 degrees. The calculated unit-cell volume of 120 nm3 is large enough to contain one protein molecule.

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.