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A procedure for the isolation of highly purified bacterial photosynthetic membranes from Rhodopseudomonas viridis is described. The purity of the final membrane fraction has been confirmed by electron microscopy. Seven major polypeptide bands are associated with the photosynthetic membranes, and all seven are resistant to solubilization in Triton X-100 detergent. Two pigmented bands with apparent molecular weights of 44K and 41K are thought to be cytochromes. The three polypeptides with apparent molecular weights of 38K, 32K, and 28K have been reported in reaction center preparations of other laboratories. Two low-molecular-weight (16K and 11K) bands bind bacteriochlorophyll b and may represent light-harvesting bacteriochlorophyll-protein complexes. The structures that were isolated seem to represent complete photosynthetic membranes, consisting of reaction center, electron transport, and light-harvesting components, all arranged in the regular lattice characteristic of viridis. Selective proteolysis of these membranes indicates that all membrane components are accessible to digestion by trypsin and pronase, except for the light-harvesting complexes.

Photosynthetic reaction centers from the bacterium Rhodopseudomonas viridis were prepared after detergent solubilization of photosynthetic membranes. The purified reaction centers, in agreement with reports from other laboratories, contain four distinct polypeptides ranging in molecular weight from 28,000 to 41,000. When the detergent was gradually removed by dialysis under appropriate conditions, large two-dimensional sheets of reaction centers were formed, suitable for analysis by electron microscopy. The crystals were rectangular, and the dimensions of a single unit cell were 121 X 129 A. Each unit cell contained four distinct subunits, each with approximate dimensions of 45 X 60 A. The thickness of the sheet was 60 A. Preliminary studies of the sheets with negative staining indicated that the sheets show a high degree of order: as many as six orders are visible in transforms of the images. Because of the fact that in R. viridis the native membrane from which these reaction centers were purified also displays a crystal-like structure, comparative studies between a membrane and one of its components, each analyzed by Fourier techniques, are now possible.

The organization of photosynthetic membranes in the cytoplasm of the photosynthetic bacterium Rh. viridis has been examined by several techniques for electron microscopy. Thin sections of membrane stacks show that the regular lattice of membrane subunits reported in other studies can be observed in thin section. Tilting of sections in the electron microscope shows that the regular lattices of several membranes overlap in a way that suggests they are in register with each other. This observation can be confirmed by freeze-fracture images in which a regular arrangement of membrane lattices can be observed, each perfectly aligned. Analysis of the spacings of membrane pairs shows that the photosynthetic membranes of Rh. viridis are very closely apposed. The mean diameter of two membranes is 160A, and the average space between two such membranes is only 42A. When a recently developed atomic level model of Rh. viridis reaction center is superimposed against these spacings, each reaction center extends from the surface of its respective membrane far enough to make contact with an apposing membrane. The limited free space between membranes and regular alignment of lattices has a number of implications for how this membrane is organized to carry out the process of energy transfer.

Because estimates of size and shape for membrane proteins are difficult to obtain directly, many workers have incorporated purified proteins into artificial lipid bilayers. Freeze-fracturing has then been used to provide a measure of the approximate size and shape of the membrane protein. We have formed reconstituted membranes containing the photosynthetic reaction center of Rhodopseudomonas viridis, a photosynthetic bacterium. The size and shape of this reaction center is accurately known from studies of negatively stained crystals of the protein to be approximately 4.5 X 6.0 nm. Freeze-fracture images of the reaction center in phosphatidyl choline liposomes show particles formed after reconstitution with an average diameter of 12.3 nm, much larger than the actual size of the protein. Deep-etched images of the surfaces of the liposomes, in which each individual reaction center complex can be seen clearly, show why the diameter of the protein is exaggerated in freeze-fracture. The individual reaction centers tend to cluster into large groups, allowing several individual reaction centers to be visualized as a single (much larger) particle in freeze-fracture. Freeze-fracturing, although a valuable tool in the analysis of membrane structure in natural and artificial membranes, must be used with caution in the estimation of molecular sizes and shapes.

The photosynthetic membranes of the purple bacterium Rhodopseudomonas viridis are composed of a semi-crystalline lattice of subunits. Proteolysis of isolated membranes with trypsin or pronase results in the degradation of polypeptides associated with the photosynthetic reaction center. However, two low molecular weight peptides which may form the light-harvesting complex survive the enzymatic treatment. The proteolysis does not affect the major absorbance peak (830 nm) associated with the reaction center. However, treatment of proteolyzed membranes with detergents such as LDAO abolishes the 830 nm absorbance peak. The 830 nm peak is stable following LDAO solubilization of non-proteolyzed membranes. These results suggest that a combination of covalent and non-covalent interactions are important in maintaining the configuration of the reaction center, and are consistent with a model of membrane organization in which the light-harvesting components are buried in a lipid phase of the membrane and reaction center components form the large structures which electron microscope studies have shown to extend from either membrane surface.