Two-dimensional structure of the native light-harvesting complex LH2 from Rubrivivax gelatinosus and of a truncated form.

The light-harvesting complex LH2 of Rubrivivax gelatinosus has an oligomeric structure built from alpha-beta heterodimers containing three bacteriochlorophylls and one carotenoid each. The alpha subunit (71 residues) presents a C-terminal hydrophobic extension (residues 51-71) which is prone to attack by an endogenous protease. This extension can also be cleaved by a mild thermolysin treatment, as demonstrated by electrophoresis and by matrix-assisted laser desorption-time of flight mass spectrometry. This cleavage does not affect the pigment binding sites as shown by absorption spectroscopy. Electron microscopy was used to investigate the structures of the native and thermolysin cleaved forms of the complexes. Two-dimensional crystals of the reconstituted complexes were examined after negative staining and cryomicroscopy. Projection maps at 10 A resolution were calculated, demonstrating the nonameric ring-like organization of alpha-beta subunits. The cleaved form presents the same structural features. We conclude that the LH2 complex is structurally homologous to the Rhodopseudomonas acidophila LH2. The hydrophobic C-terminal extension does not fold back in the membrane, but lays out on the periplasmic surface of the complex.

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.

Use of detergents in two-dimensional crystallization of membrane proteins.

Structure determination at high resolution is actually a difficult challenge for membrane proteins and the number of membrane proteins that have been crystallized is still small and far behind that of soluble proteins. Because of their amphiphilic character, membrane proteins need to be isolated, purified and crystallized in detergent solutions. This makes it difficult to grow the well-ordered three-dimensional crystals that are required for high resolution structure analysis by X-ray crystallography. In this difficult context, growing crystals confined to two dimensions (2D crystals) and their structural analysis by electron crystallography has opened a new way to solve the structure of membrane proteins. However, 2D crystallization is one of the major bottlenecks in the structural studies of membrane proteins. Advances in our understanding of the interaction between proteins, lipids and detergents as well as development and improvement of new strategies will facilitate the success rate of 2D crystallization. This review deals with the various available strategies for obtaining 2D crystals from detergent-solubilized intrinsic membrane proteins. It gives an overview of the methods that have been applied and gives details and suggestions of the physical processes leading to the formation of the ordered arrays which may be of help for getting more proteins crystallized in a form suitable for high resolution structural analysis by electron crystallography.

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.

Head direction cells: properties and functional significance.

The strong signal carried by head direction cells in the postsubiculum complements the positional signal carried by hippocampal place cells; together, the directional and positional signals provide the information necessary to permit rats to generate and carry out intelligent, efficient solutions to spatial problems. Our opinion is that the hippocampal positional system acts as a cognitive map and that the role of the directional system is to put the map into register with the environment. In this way, paths found using the map can be properly executed. Head direction cells have recently been discovered in parts of the thalamus reciprocally connected with the postsubiculum; such cells provide important clues to the organization of the directional system.

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.

The positional firing properties of medial entorhinal neurons: description and comparison with hippocampal place cells.

Hippocampal place cells in the rat are so named because they fire predominantly within circumscribed regions of the environment. This study describes the positional firing properties of cells afferent to hippocampal place cells, in superficial layers of medial entorhinal cortex (MEC). MEC cells in these layers project to the hippocampus via the perforant path and, along with lateral entorhinal cells, are the sole route by which cortical information reaches the hippocampus. MEC cells were recorded from rats while they retrieved pellets in simple geometric enclosures. The behavioral task as well as procedures for data collection and analysis were the same used in previous studies on hippocampal place cells (e.g., Muller et al., 1987) in order to facilitate the direct comparison between hippocampal and entorhinal cells. The firing patterns of MEC cells show pronounced locational variations reminiscent of hippocampal firing fields, but with a lower signal-to-noise ratio. While noisy, MEC firing patterns are stationary in time as evidenced by their reproducibility, and the improvement in spatial signal with long-duration recordings. Furthermore, MEC firing patterns are not due to variations in the rat's behavior. Taken together, these data show that the positional firing variations in MEC cells are due to the location-specificity of MEC cells. These and additional data lead us to conclude that location-specific information exists prior to the hippocampus. MEC cells are similar to hippocampal place cells in that their firing can be controlled by the rotation of a visual cue (a white card attached to the wall), but is not disrupted by removing the cue. An important difference between hippocampal and entorhinal cells was seen when the shape of the recording chamber was changed. In the transition from a cylinder to an equal-area square of similar appearance, MEC firing patterns topologically transformed (or "stretched") while those of hippocampal place cells changed to an unpredictable pattern. We conclude that the positional firing of MEC cells is more "sensory bound" than hippocampal cells, and that the ability to discriminate different environments, while present in the hippocampus, is not yet present in its input from MEC.

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.

Head-direction cells recorded from the postsubiculum in freely moving rats. I. Description and quantitative analysis.

This paper is a study of the behavioral and spatial firing correlates of neurons in the rat postsubiculum. Recordings were made from postsubicular neurons as rats moved freely throughout a cylindrical chamber, where the major cue for orientation was a white card taped to the inside wall. An automatic video/computer system monitored cell discharge while simultaneously tracking the position of 2 colored light emitting diodes (LEDs) secured to the animal's head. The animal's location was calculated from the position of one of the LEDs and head direction in the horizontal plane calculated from the relative positions of the 2 LEDs. Approximately 26% of the cells were classified as head-direction cells because they discharged as a function of the animal's head direction in the horizontal plane, independent of the animal's behavior, location, or trunk position. For each head-direction cell, vectors drawn in the direction of maximal firing were parallel throughout the recording chamber and did not converge toward a single point. Plots of firing rate versus head direction showed that each firing-rate/head-direction function was adequately described by a triangular function. Each cell's maximum firing rate occurred at only one (the preferred) head direction; firing rates at head directions on either side of the preferred direction decreased linearly with angular deviation from the preferred direction. Results from 24 head-direction cells in 7 animals showed an equal distribution of preferred firing directions over a 360 degrees angle. The peak firing rate of head-direction cells varied from 5 to 115 spikes/sec (mean: 35). The range of head-direction angles over which discharge was elevated (directional firing range) was usually about 90 degrees, with little, if any, discharge at head directions outside this range. Quantitative analysis showed the location of the animal within the cylinder had minimal effect on directional cell firing. For each head-direction cell, the preferred direction, peak firing rate, and directional firing range remained stable for days. These results identify a new cell type that signals the animal's head direction in its environment.

Head-direction cells recorded from the postsubiculum in freely moving rats. II. Effects of environmental manipulations.

The discharge characteristics of postsubicular head-direction cells in a fixed environment were described in the previous paper (Taube et al., 1990). This paper reports changes in the firing properties of head-direction cells following changes in the animal's environment. Head-direction cells were recorded from rats as they moved freely in a 76-cm-diameter gray cylinder. A white card, occupying 100 degrees of arc, was taped to the inside wall of the cylinder and served as the major orienting spatial cue in the animal's environment. Rotation of the cue card produced near-equal rotation in the preferred firing direction of head-direction cells, with minimal changes in peak firing rate, directional firing range, or asymmetry of the firing-rate/head-direction function. Card removal had no effect on peak firing rate or range of firing, but in 8/13 cells the preferred direction rotated by at least 24 degrees. Similarly, changing the shape of the environment to a rectangular or square enclosure caused the preferred firing direction to rotate by at least 48 degrees for 8/10 cells in the rectangle and 3/8 cells in the square, with minimal changes in the peak firing rate or directional firing range. Hand holding the animals and moving them around the cylinder had no effect on the preferred direction or firing range of the cell, but decreased the maximal firing rate in 7/9 cells. On 2 occasions, 2 head-direction cells were recorded simultaneously. The rotation of the preferred firing direction for one cell was the same as the rotation of the preferred direction for the second cell after each environmental manipulation. These results demonstrate that specific visual cues in the environment can exert control over the preferred firing direction and indicate that head-direction cell firing is not a simple sensory response to visual cues, but rather represents more abstract information concerning the animal's spatial relationship with its environment. The constancy of the angle between the preferred firing directions of pairs of simultaneously recorded head-direction cells suggests that there is a fixed mapping of the population onto direction within the environment. Thus, environmental manipulations appear to cause only a change in the reference direction, but leave all other discharge characteristics of directional cells unchanged. In the discussion, comparisons are drawn between the responses of head-direction cells and hippocampal place cells to similar environmental manipulations (Muller and Kubie, 1987), and ways in which these 2 spatial systems interact in navigation are discussed.(ABSTRACT TRUNCATED AT 400 WORDS)

Spatial firing patterns of hippocampal complex-spike cells in a fixed environment.

A TV/computer technique was used to simultaneously track a rat's position in a simple apparatus and record the firing of single hippocampal complex-spike neurons. The primary finding is that many of these neurons behave as "place cells," as first described by O'Keefe and Dostrovsky (1971) and O'Keefe (1976). Each place cell fires rapidly only when the rat is in a delimited portion of the apparatus (the cell's "firing field"). In agreement with O'Keefe (1976) and many other authors, we have seen that the firing of place cells is highly correlated with the animal's position and is remarkably independent of other aspects of the animal's behavioral state. Several properties of firing fields were characterized. Firing fields are stable over long time intervals (days) if the environment is constant. They come in several shapes when the animal is in a cylindrical apparatus; moreover, the set of field shapes is different when the animal is in a rectangular apparatus. It also seems that a single cell may have more than one field in a given apparatus. By collecting a sample of 40 place cells in a fixed environment, it has been possible to describe certain features of the place cell population, including the spatial distribution of fields within the apparatus, the average size of fields, and the "intensity" of fields (as measured by maximum firing rate). We also tested the hypothesis that the firing rate of each place cell signals the animal's distance from a point (the field center) so that a weighted average of the firing of the individual cells encodes the animal's position within the apparatus. The animal's position, calculated according to this "distance hypothesis," is systematically different from the animal's true position; this implies that the hypothesis in its simplest form is wrong.