The proposed research will improve methods for high-resolution electron microscopy of macromolecules, and will pursue applications of these to cogen biological problems. Coating by thin tungsten films effectively contrasts 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 films which will fully exploit available resolving power, and which will be suited to the inherently high-contrast darkfield imaging mode. To this end we will explore use of mixtures of refractory metals (which should limit possibilities for crystalization and thus reduce crystallite size) and of low-temperature specimens 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 hamster female protein, IgM from invertebrates, human lubricin, bronchial mucins etc. We plan applications of the electronmicroscopic mapping approach, in which macromolecules are complexed with readily-resolvable, site-specific markers to a number of problems. Chemically-defined sites on the fibrinogen molecule, including the important region which binds factor XIII, staphylococcin & 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. Skeletal and cardiac myosin will be mapped with specific Fabs directed against subregions of these molecules (S1, S2, light chains & 50 kd & 25kd S1 subdomains) in order to elucidate topography of the myosin head region. Positions of carbohydrate residues along epiglycanin (a mouse mammary tumor cell-surface glycoprotein) will be mapped by a series of specific lectins, and by rabbit & monoclonal IgG & 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. We will characterize cardiac myosin in a combined physical-chemical and electron microscopic study, as well as assessing the effects of light chain removal on function and structure of myosin and its polymeric forms.
Showing the most recent 10 out of 20 publications
