This application is concerned exclusively with determining the structure of photosystem II, a photosynthetic reaction center found in eukaryotic organisms. Photosystem II is unique in that it is able to split water, evolving oxygen in the process. consequently, it is of prime importance to the biosphere. Obviously, the complex itself is not directly related to any human disease, but this is basic research on a very important membrane complex whose function has very direct effects on air quality. The work will involve cryoelectron microscopy, primarily on two- dimensional crystals of photosystem II. the methodology for causing these crystals to form has already been worked out and the crystals have yielded 1.7 nm resolution on negatively stained samples. The projection map from the negatively stained samples has already shown several important features, including a density which extends quite far from the main body of the complex and may be involved in interactions with other complexes. In addition, it appears that two PS II complexes are found in close opposition, with the two reaction center cores approximately 0.8 nm apart. During the course of the proposed project, a complete characterization of the crystals will be done, including immunoblots with each of the polypeptides found in the PS II complex. Most importantly, the preparations will be examined for biological activity, so that structure can be related to function. A three-dimensional reconstruction, using frozen hydrated samples, will be done during the course of the project. The use of cryoelectron microscopy should improve the resolution which is obtainable. However, the major advantage of cryoelectron microscopy is that the complexes will be in a physiological buffer, with no heavy metals or stain to obscure details or disrupt function. Also, that portion of the complex buried in the membrane can be visualized with frozen hydrated samples, so that a reconstruction will include the complete complex. In addition, individual polypeptides will be localized on projection maps, using antibody labeling and image processing. The location of the polypeptides, as determined from the antibody work, will then be related to three-dimensional structure. the crystallization process results in the loss of three polypeptides involved in oxygen evolution. These polypeptides will be reconstituted, one-by-one, and their locations determined on projection maps. The results will provide a complete picture of the structure of photosystem II.
