The proposed research will improve methods for high-resolution EM of individual macromolecules, and will pursue applications of these to cogent biological problems. Coating by thin refractory metal films effectively contrasts individual macromolecular substructure. Due to recent improvements in electron optics, grain size, rather than instrumental factors, currently limits ultimate resolution. Thus, we intend to produce fine-grain coatings which will fully exploit available resolving power, particularly applying high-contrast darkfield imaging. We will explore use of mixtures of refractory metals (which should limit possibilities for crystallization and thus reduce crystallite size) and of low temperature specimen supports (which should favor film continuity). Effects of solvent systems and drying conditions will also be studied. These techniques will be applied to a number of macromolecular systems, including myosin, fibrinogen, epiglycanin, retinol-binding protein, antigen-antibody complexes, bronchial mucins, and hamster female protein. We plan applications of the electronmicroscopic mapping approach, in which macromolecules are complexed with readily-resolvable, site-specific protein markers, to a number of problems. Chemically-defined sites in the fibrinogen molecule, including the important region which binds factor XIII, staphylococcin and blood platelets, will be located by complexing with specifically-directed Fabs or with monoclonal antibodies. Positions of the 4 carbohydrate residues in this molecule will be determined by complexing with specific lectins. We will further characterize cardiac myosin in a combined physical-chemical and electron microscopic study, as well as assessing the effects of various physiological conditions on function and structure of myosin S1 region, and polymeric forms. Cardiac myosin will be mapped with specific Fabs directed against subregions of these molecules (S1, S2, light chains, S1 subdomains and hinge) in order to elucidate topography of the myosin head region. Positions of specific epitopes and carbohydrate residues along epiglycanin (a mouse mammary tumor cell-surface glycoprotein) will be mapped by a series of specific lectins, and by rabbit and monoclonal IgG and IgM. The latter will be used to determine the specificities of these antibodies for epiglycanin, and for the monospecific site, respectively. Certain other large glycoproteins will also be mapped.
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