Kenneth R. Miller - US grants

The molecular organization of the photosynthetic membrane and its components will be investigated by high resolution electron microscope techniques. Three systems will be used for study: a bacterial photosynthetic membrane, photosynthetic membranes from higher plants in which ordered lattices of membrane subunits have been induced, and a reconstituted system containing a chlorophyll-protein complex in an artificial lipid bilayer. Each of these systems has shown a high degree of two-dimensional order, and this characteristic will be exploited in analyzing their detailed substructure. Electron and optical diffraction techniques, combined with computer analysis of electron micrographs, will be used to form high resolution synthesized images of the membrane systems. It is expected that these high resolution maps will provide insights into the nature of the photosynthetic process and the structure of biological membranes in general.

The aim of this research project is to develop a more complete understanding of the organization, function, and composition of the photosynthetic membrane. The organism which will be used for these studies is Rhodopseudomonas viridis, a photosynthetic bacterium. The photosynthetic membranes of Rh. viridis are ideal for these investigations for two reasons: they are relatively simple biochemically (containing no more than 7 polypeptides in the membrane), and the membranes are organized in a regular crystalline pattern, making it possible to use image processing techniques to obtain high resolution information from electron micrographs. In addition, a growing body of work on Rh. viridis, including a high resolution map of the membrane's reaction center complex, will allow our work to move rapidly towards a complete understanding of the membrane. Electron microscopy will be used to prepare high resolution maps of the membrane under a variety of conditions. Low-dose electron microscopy will be used to reduce electron beam damage, media of differing contrast will help us to define protein and lipid boundries within the membrane, and low-temperature microscopy will allow the direct observation of hydrated membranes. Images prepared in these ways will be analyzed by Fourier techniques, as well as by the more recently developed correlation systems, and a high resolution image of the membrane will emerge. These studies will be supplemented by direct mass estimates of the membrane made by Scanning Transmission Electron Microscopy (STEM), and images reconstructed from stacks of the membrane in intact cells. Biochemical studies on the membrane will supplement the structural work. The arrangements of polypeptides in the membrane will be studied by chemical crosslinking of the membrane. Light-harvesting bacteriochlorophyll-protein complexes will be isolated and studied, and attempts will be made to crystallize light-harvesting complexes so that their structure can be analyzed as well. Antibodies against specific regions of the light-harvesting complexes will be prepared and used to localize their positions within the high-resolution EM map of the membrane. Finally, attempts will be made to discover the means by which the Rh. viridis photosynthetic membrane transduces energy into chemical terms. We will search for a membrane-associated ATP-synthetase, something that has thus far eluded workers in this system. Our experiments to find this ATPase will be biochemical and immunological, and if the search is successful, the ATP-synthetase will be localized at the level of electron microscopy. Although photosynthetic membranes are not directly related to health research, the Rh. viridis photosynthetic membrane is in fact an idea model system in which the organization of biological membranes and their function in the important process of energy transduction can be effectively investigated.

scanning electron microscopy; biomedical equipment purchase;