DEVELOPMENT OF LOW TEMPERATURE AFM, FREEZE FRACTURE/ETCH

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Elucidation of protein conformational changes and their interactions, along with higher order structures, has been important and exciting goal of structural biology research. However, a generally applicable technique for determining the structures of non-crystalline macromolecules at high resolution (sub-nanometer range) has not been developed. Atomic force microscopy does not require a thin or a crystalline specimen, and has the potential of atomic resolution, limited only by deformation of soft materials. The main purpose of this project is to develop an atomic force microscope for operation at liquid nitrogen temperature. Combination of such a lower temperature atomic force microscope with well established freeze fracture and freeze etching techniques will permit the study of fine structures of membrane- associated macromolecules, stabilized against deformation at low temperatures, at resolutions better than 1 nm. With the help of different specimen preparatory techniques, even purified non-lip reconstituted macromolecules can be studied in such a system. In principle, no crystallization and/or specimen manipulation, such as fixation, straining or making replica, are required in this technique. The three year project period will be utilized to design and construct a liquid nitrogen temperature atomic force microscope operating at ambient pressure. This is a novel approach, preferred for its relatively easy implementation (compared with UHV based systems) and providing a super clean working environment. The complete system, including the ambient pressure freeze fracture/freeze etching apparatus operated in liquid nitrogen (fracture) and/or in pure nitrogen vapor (fracture and etching), will be housed in a well insulated dewar with a baffle assembly to eliminate water contamination through diffusion. The low temperature part is remotely operable to reduce specimen contamination and to speed up specimen exchange. Other imaging methods, such as confocal microscopy and electron microscopy, will be used extensively as supplementary techniques, in order to assure that the instrument performs with minimal artifacts. Successful implementation of this project will provide a unique capability for the study of the structure of macromolecules, which cannot be accomplished with other currently available techniques, such as electron microscopy and X-ray diffraction techniques, and will contribute to our understanding of many important biological processes and mechanism based on conformational changes in macromolecules.