The purpose of this Project is to develop suitable methodology for the newly invented atomic force microscope [AFM], so that, high resolution images of membrane associated macromolecules can be obtained at native or near native conditions. Specifically, we will further develop the instrument itself for biomedical research, to develop and supported Langmuir-Blodgett films as a general specimen preparatory techniques, and also to study the molecular structures and conformations of two ATP-driven ion pumps: Ca2+-ATPase and Na+/K+-ATPase, important in cardiovascular systems. As well known, the application of atomic force microscopy to biological structural determination has become handicapped by two major obstacles: (1) the large probing force (100nN - 0.1nN) that deforms and damages the specimen structures and (2) the poor specimen stability and adhesion to the substrate, as well as the difficulty to find the areas of interest and a specimen. The instrumentation proposed in this Project will incorporate a high-powered light microscope to a fluid cell that has a built-in motion stage. Such an approach is aimed to reduce the difficulty in operation and to aid the localization of the specimen buffer solution. The use of fluid cell also enables us to operate the atomic force microscope at 0.1nN range, due to the removal of surface tension. The supported Langmuir-Blodgett lipid film will solve the problem of specimen adhesion to the substrate and provide the opportunity of using cross- linkers to further improve mechanical stability. When protein-enriched vesicles are fused with such a lipid monolayer, a near uniform, large specimen can be made, which also simplifies specimen searching. Based on the published results on supported lipid membranes, when the technique developed is applied to ATPases, we expect 1 nm resolution achievable that may be sufficient to study conformational changes at various conditions, easily achievable in the fluid cell via solution exchanges. The instrument development is a necessary step toward a user-friendly dedicated atomic force microscope for biomedical science and the specimen preparation via supported Langmuir-Blodgett lipid films in a generally applicable technique for all membrane associated macromolecules. The development of specimen preparation methods for biological atomic force microscope is as important as the instrument itself. Our long-term goal is to develop various scanning probe microscopies, such as ionic surface scanning tunneling and cryo-atomic force for structural biology applications, and to use these high resolution techniques to elucidate molecular structures and conformations of membrane associate proteins.
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