High-resolution AFM topographs of Rubrivivax gelatinosus light-harvesting complex LH2.

Light-harvesting complexes 2 (LH2) are the accessory antenna proteins in the bacterial photosynthetic apparatus and are built up of alphabeta-heterodimers containing three bacteriochlorophylls and one carotenoid each. We have used atomic force microscopy (AFM) to investigate reconstituted LH2 from Rubrivivax gelatinosus, which has a C-terminal hydrophobic extension of 21 amino acids on the alpha-subunit. High-resolution topographs revealed a nonameric organization of the regularly packed cylindrical complexes incorporated into the membrane in both orientations. Native LH2 showed one surface which protruded by approximately 6 A and one that protruded by approximately 14 A from the membrane. Topographs of samples reconstituted with thermolysin-digested LH2 revealed a height reduction of the strongly protruding surface to approximately 9 A, and a change of its surface appearance. These results suggested that the alpha-subunit of R.gelatinosus comprises a single transmembrane helix and an extrinsic C-terminus, and allowed the periplasmic surface to be assigned. Occasionally, large rings ( approximately 120 A diameter) surrounded by LH2 rings were observed. Their diameter and appearance suggest the large rings to be LH1 complexes.

Use of octyl beta-thioglucopyranoside in two-dimensional crystallization of membrane proteins.

A great interest exists in producing and/or improving two-dimensional (2D) crystals of membrane proteins amenable to structural analysis by electron crystallography. Here we report on the use of the detergent n-octyl beta-d-thioglucopyranoside in 2D crystallization trials of membrane proteins with radically different structures including FhuA from the outer membrane of Escherichia coli, light-harvesting complex II from Rubrivivax gelatinosus, and Photosystem I from cyanobacterium Synechococcus sp. We have analyzed by electron microscopy the structures reconstituted after detergent removal from lipid-detergent or lipid-protein-detergent micellar solutions containing either only n-octyl beta-d-thioglucopyranoside or n-octyl beta-d-thioglucopyranoside in combination with other detergents commonly used in membrane protein biochemistry. This allowed the definition of experimental conditions in which the use of n-octyl beta-d-thioglucopyranoside could induce a considerable increase in the size of reconstituted membrane structures, up to several micrometers. An other important feature was that, in addition to reconstitution of membrane proteins into large bilayered structures, this thioglycosylated detergent also was revealed to be efficient in crystallization trials, allowing the proteins to be analyzed in large coherent two-dimensional arrays. Thus, inclusion of n-octyl beta-d-thioglucopyranoside in 2D crystallization trials appears to be a promising method for the production of large and coherent 2D crystals that will be valuable for structural analysis by electron crystallography and atomic force microscopy.

Supramolecular organization of the photosynthetic apparatus of Rhodobacter sphaeroides.

Native tubular membranes were purified from the purple non-sulfur bacterium Rhodobacter sphaeroides. These tubular structures contain all the membrane components of the photosynthetic apparatus, in the relative ratio of one cytochrome bc1 complex to two reaction centers, and approximately 24 bacteriochlorophyll molecules per reaction center. Electron micrographs of negative-stained membranes diffract up to 25 A and allow the calculation of a projection map at 20 A. The unit cell (a = 198 A, b = 120 A and gamma = 103 degrees) contains an elongated S-shaped supercomplex presenting a pseudo-2-fold symmetry. Comparison with density maps of isolated reaction center and light-harvesting complexes allowed interpretation of the projection map. Each supercomplex is composed of light-harvesting 1 complexes that take the form of two C-shaped structures of approximately 112 A in external diameter, facing each other on the open side and enclosing the two reaction centers. The remaining positive density is tentatively attributed to one cytochrome bc1 complex. These features shed new light on the association of the reaction center and the light-harvesting complexes. In particular, the organization of the light-harvesting complexes in C-shaped structures ensures an efficient exchange of ubihydroquinone/ubiquinone between the reaction center and the cytochrome bc1 complex.

A new "gel-like" phase in dodecyl maltoside-lipid mixtures: implications in solubilization and reconstitution studies.

The interaction of dodecyl maltoside with lipids was investigated through the studies of solubilization and reconstitution processes. The solubilization of large unilamellar liposomes was analyzed through changes in turbidity and cryo-transmission electron microscopy. Solubilization was well described by the three-stage model previously reported for other detergents, and the critical detergent/phospholipid ratios at which lamellar-to-micellar transition occurred (Rsat = 1 mol/mol) and finished (Rsol = 1.6 mol/mol) were determined. The vesicle-micelle transition was further observed in the vitrified hydrated state by cryo-transmission electron microscopy. A striking feature of the solubilization process by dodecyl maltoside was the discovery of a new phase consisting of a very viscous "gel-like" sample. It is shown that this equilibrium cohesive phase is composed of long filamentous thread-like micelles, over microns in length. Similar structures were observed upon solubilization of sonicated liposomes, multilamellar liposomes, or biological Ca2+ ATPase membranes. This "gel-like" phase was also visualized during the process of liposome reconstitution after detergent removal from lipid-dodecyl maltoside micelles. The rate of detergent removal, controlled through the use of SM2 Bio-Beads, was demonstrated to drastically influence the morphology of reconstituted liposomes with a propensity for multilamellar liposome formation upon slow transition through the "gel-like" phase. Finally, on the basis of these observations, the mechanisms of dodecyl maltoside-mediated reconstitution of bacteriorhodopsin were analyzed, and optimal conditions for reconstitution were defined.

Bio-Beads: an efficient strategy for two-dimensional crystallization of membrane proteins.

This work establishes the potential of Bio-Beads as a simple alternative to conventional dialysis for removing detergent and for obtaining 2D crystals of integral membrane proteins useful for structure analysis by electron crystallography. Kinetic and equilibrium aspects of removal of different detergents by adsorption onto hydrophobic Bio-Beads SM2 have been systematically investigated and extended to 2D crystallization of different prototypic membrane proteins, including: (a) Ca2+ ATPase from sarcoplasmic reticulum; (b) melibiose permease from Escherichia coli; (c) cytochrome b6f from Chlamydomonas reinhardtii. Different crystals could be produced from all protein preparations, with optical diffraction down to 20-25 A in negative stain.

Electron diffraction of helical particles.

The development of low-dose electron cryo-microscopy has provided the means to see structural details to better than 10 A resolution in helical structures. The application of techniques of image analysis to micrographs can yield accurate phases, but not amplitudes with which to generate three-dimensional maps of the structure. Electron diffraction can provide reliable amplitudes, which can be combined with the phases from the images. In order to collect amplitude data, two problems have to be overcome: the pattern should be obtained from a large well ordered sample of particles, and the inelastic background should be properly subtracted. In this paper, we present three simple methods to produce rafts of helical particles. Using these methods we have obtained electron diffraction patterns from TMV (with data out to 0.28 nm), TMV protein stacked disks (with data out to 0.3 nm) and bacterial flagellar filaments (with data out to 0.5 nm). In addition, we describe the algorithms used to extract the amplitudes from the diffraction patterns.

Small angle X-ray scattering and electron cryomicroscopy study of actin filaments: role of the bound nucleotide in the structure of F-actin.

Actin filaments (F-actin) complexed to various nucleotides (ADP (adenosine diphosphate) ADP-P(i) (P(i), inorganic phosphate), and ADP-BeF3- (BeF3-, beryllium fluoride)) have been studied by small angle X-ray scattering and electron cryomicroscopy. The small angle X-ray scattering data show that the value of the cross-radius of gyration (2.55 +/- 0.15 nm) and the mean helical symmetry of the filaments are independent of the nature of the bound nucleotide. The scattering profiles of all the F-actin suspensions exhibit secondary maximal located at similar positions. However, the intensities of the secondary maxima depend upon both the protein concentration and the type of nucleotide bound to the F-actin. These variations indicate that F-actin has different properties (such as its tendency to aggregate and the structure of the filament), which depend upon the type of nucleotide bound. Electron cryomicroscopy of vitrified suspensions shows that F-actin may have different conformations. The nonequatorial layer lines, which constitute the Fourier transforms of the filament images, have different signal to noise ratios depending on the type of nucleotide bound to the actin. The 3-D reconstructions of the actin filaments show an inner and outer domain. In the maps, the visibility of the outer domain depends upon the type of nucleotide bound to the actin. It respectively increases for ADP-P(i), ADP, and ADP-BeF3-. We attribute these differences to the disorder of the filaments, which in turn depends upon the nucleotide bound. These variations in disorder imply that there are structural changes (contacts, structure, or orientation) in the actin subunits forming the filaments. These changes may have an important role in the motility processes in which actin is involved.

Electron cryomicroscopy of frozen-hydrated biological specimens: analysis of freezing artifacts by X-ray cryocrystallography.

Freezing artifacts have been evaluated by X-ray cryocrystallography on pellets of two-dimensional membrane protein crystals: purple membrane and maltoporin. The comparison of the X-ray patterns recorded when the specimens are maintained at room temperature to those obtained when the specimens are maintained at about -160 degrees C shows that (i) membrane proteins have a positive thermal dilatation coefficient: the protein crystal lattice shrinks upon cooling; (ii) the asymmetric unit of crystal containing water is changed upon freezing; the relative intensities of the diffraction rings of such crystals are different after freezing. From these results, it can be postulated that freezing may lead to partial dehydration of biological objects. Electron cryomicroscopy visualizes objects which are structurally influenced by the cooling procedure. However, our microscopy study on maltoporin crystals shows that freezing artifacts are negligible in comparison to artifacts associated with conventional techniques such as negative staining.

Cryo-electron microscopy of vitrified specimens: an approach to the study of bulk specimens.

We are using and developing cryo-electron microscopy of vitrified specimens. Our main interests concern the structure of muscle and muscular components. Micrographs which generally contain periodic features are analyzed by numerical image processing methods. To detect artifacts induced by the electron microscopy techniques, we correlate our results to those obtained by X-ray diffraction. In this paper, we describe our approach to the study of bulk specimens. Vitrification of such specimens is assessed by cryo-X-ray diffraction. Microscopy is done on cryo-substituted specimens.

Time-resolved cryo-electron microscopy of vitrified muscular components.

Biological objects may be arrested in defined stages of their activity by fast freezing and may then be structurally examined. If the time between the start of activity and freezing is controlled, structural rearrangements due to biological function can be determined. Cryo-electron microscopy shows great potential for the study of such time-dependent phenomena. This study examines the actin polymerization process using cryo-electron microscopy of vitrified specimens. Actin filaments are shown to undergo a structural change during polymerization. In the early stages of the polymerization process (t less than 2 min), filaments exhibit a pronounced structural variation and frequently show a central low-density area. In the later stages of the polymerization, F-actin-ADP filaments have a more uniform appearance and rarely display a central low-density area. These findings, analysed on the basis of a previously proposed polymerization model, suggest that polymerization intermediates (F-actin-ATP and more probably F-actin-ADP-Pi) and filaments at steady state (F-actin-ADP) have different structures. To investigate the physiological relevance of these results at the cellular level, the potential of cryo-substitution in preserving the structure of muscular fibre was assessed. Optical diffraction patterns of relaxed and contracted frog cutaneous muscle are similar to the corresponding X-ray diffraction patterns. The resolution of the images extends to about 7 nm. These results show that dynamic study of muscle contraction is possible using cryo-substitution.

Purification of the temperature-specific surface antigen of Paramecium primaurelia with its glycosyl-phosphatidylinositol membrane anchor.

The membrane form of the temperature-specific G surface antigen of Paramecium primaurelia strain 156 has been purified by a novel procedure utilizing solubilization by detergent, ammonium sulfate precipitation, and high-performance liquid chromatography. The surface antigen, which was prepared in a nondenatured state containing a glycosyl-phosphatidylinositol membrane anchor, migrated as a single band upon electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. Following cleavage of the purified surface antigen by a phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis, the soluble form was released with the unmasking of a particular glycosidic immunodeterminant called the cross-reacting determinant. The purification protocol described here will now permit further biochemical and biophysical characterization of the nondenatured membrane form of Paramecium surface antigens.

X-ray analysis of the kinetics of Escherichia coli lipid and membrane structural transitions.

Synchrotron radiation was used to follow the time course of the transitions, induced by temperature jump, in Escherichia coli membranes and their lipid extracts isolated from a fatty acid auxotroph grown with different fatty acids. We measured the relaxation times associated with the phase transitions as well as with the conformational transition of the hydrocarbon chains and observed different behavior as a function of chemical composition. Relaxation times of about 1-2 s were found at a hexagonal to lamellar phase transition and within a lamellar phase whose parameters display important variations with temperature when the conformational transition takes place. On the other hand, no delay was observed for a phase transition where large lipid or water diffusion was not needed. We have shown that phase transitions and conformational transitions are, to a large extent, uncoupled and that the relaxation times corresponding to the latter transition could be related to the size of the ordered domains. In all cases, the order to disorder conformational transition is more rapid than the disorder to order transition. Finally, the relaxation times of the disorder to order transition observed with the membranes and with their lipid extracts were found to be strongly correlated, indicating that the proteins do not play a role in this transition.

Polymyxin B-induced phase separation and acyl chain interdigitation in phosphatidylcholine/phosphatidylglycerol mixtures.

Monolayers, fluorescence polarization, differential scanning calorimetry and X-ray diffraction experiments have been carried out to examine the effect of the polypeptide antibiotic polymyxin B on the phase behaviour of dipalmitoylphosphatidylglycerol (DPPG) either pure or mixed with dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC). It is shown that in both phosphatidylglycerol alone and phosphatidylglycerol/phosphatidylcholine mixtures, polymyxin B can induce either phase separation between lipid domains of various compositions or interdigitation of the acyl chains in the solid state, without segregation of the two lipids. Phase separation was observed by fluorescence and differential scanning calorimetry after addition of the antibiotic to vesicles composed of mixtures of DMPC and DPPG in conditions where polymyxin B did not saturate phosphatidylglycerol (DPPG to polymyxin B molar ratio, Ri, higher than 15). Phase separation was also observed in mixed monolayers of DPPC and of the 5:1 DPPG/polymyxin B complex, at high surface pressure. Acyl chain interdigitation was observed by X-ray diffraction in both 5:1 DPPG/polymyxin B mixtures and preformed 5:5:1 DMPC/DPPG/polymyxin B mixture, in which the antibiotic saturates phosphatidylglycerol (Ri 5). In both cases, raising the temperature gave rise to a complex double-peaked phase transition by differential scanning calorimetry, from the interdigitating phase to a normal L alpha lamellar phase. As it is known that polymyxin B does not interact with phosphatidylcholine, the data presented show that, when phosphatidylcholine and phosphatidylglycerol are mixed together, a phase perturbation such as acyl chain interdigitation, which normally affects only phosphatidylglycerol, is also felt by phosphatidylcholine.

Euglena plasma membrane during normal and vitamin B12 starvation growth.

Freeze-fracture and optical diffraction techniques were used to study the organization of the Euglena pellicle during the normal and replicative stages of the cell cycle and during vitamin B12 starvation. It was shown that the diffuse layer underlying the tripartite structure has a fibrillar structure. Despite the absence of homology in the 2 fracture faces of the pellicle, the EF striated and the PF particulate ones appear complementary as shown by optical diffraction studies; it must therefore be considered as a true membrane. The grooves are free from such particles and striations. They appear as a specific pattern of the cortex, different from the ridges in their structural organization and their replicative capacity as observed during vitamin B12 starvation. This notion is confirmed by the mode of pellicular growth which is characterized by 2 steps. The first occurs during the early replicative stage (pre-mitotic phase of the cell cycle) when the formation of a new ridge is correlated with the appearance of the 'minor' orientation of a 2-dimensional lattice on the EF and the PF faces and the spread of the particles over the PF face of the space between the old ridges. The second takes place during the lengthening of the ridges from the initiating posterior side (non-replicative stage). During this second step, the 'major' orientation of the lattice is preferentially observed in control cells and exclusively in starved cells. The striking differences between the grooves and the ridges is discussed, as well as the 2 modes of growth and their significance in morphogenesis.

A novel packing of the hydrocarbon chains in lipids. The low temperature phases of dipalmitoyl phosphatidyl-glycerol.

The system dipalmitoyl phosphatidylglycerol-water displays several phases in the temperature-concentration range explored in this work. All the phases are lamellar; they differ by the organization of the hydrocarbon chains. In the high temperature phase the conformation of the chains is liquid-like. In the low temperature phases the chains are stiff and parallel and they interdigitate (in other words the CH3 ends of the chains of one layer are near to the polar groups of the opposite layer). Moreover, several types of packings of the stiff chains are observed which differ by the symmetry of the two-dimensional lattices. The observed lattices are p6, cmm and pgg.