Changes in the conformation and assembly of proteins governs most processes in cell biology. The atomic or scanning force microscope (AFM) is a new imaging modality that has powerful and unique capabilities to study these processes directly because of its high spatial resolution (atomic on hard surfaces), extended force range (up to 10-15N) sufficient to measure virtually any molecular interaction, nanoscale control, capability of being functionalized so it acts as a very specific sensor of specifici biomolecules, relative lack of destructiveness in operation and preparation, and the ability to image specimens dry and in physiologic solutions. These capabilities set AFM apart from traditional imaging modalities and open up a new approach to structural biology. This proposal brings together the expertise of Susan Lindquist's laboratory which is among the leaders in investigating cytoplasmically inherited genetic elements and the Arnsdorf Laboratory which has been developing the AFM for biomedical research. Among the most interesting cytoplasmically inherited genetic elements are the infective prison proteins that cause the transmissible spongiform encephalopathies such as """"""""mad cow disease"""""""" in cattle and a variety of progressively lethal neurodegenerative diseases in man. The prion protein is normally found in the cell membrane, and recent investigations support the hypothesis that conformationally abnormal forms of this protein are the infectious particles responsible for these diseases. Two cytoplasmically inherited genetic elements in yeast propagate by a similar mechanism producing heritable changes in translational fidelity or nitrogen metabolism. [PSI+] is the best characterized yeast prion and is believed to be a self-perpetuating conformational alteration in the nuclear-encoded Sup35 protein, a subunit of the translation-termination apparatus. Propagation of the [PSI+] element depends on a specific concentration of heat- shock protein 104 (Hsp 104) which acts as a chaperone, but somewhat paradoxically overexpression of HSP104 can """"""""cure"""""""" cells of the [PSI+] element and restore normal translational fidelity. We will investigate with the AFM: (1) the surface structure of Sup35, its component subunit """"""""building blocks"""""""", possible intermediates, and aggregates; (2) the time course of (1); (3) the self-perpetuating, alternative modes of subunit polymerization and its relationship to strain variation in [PSI+]; (4) the forces of interactions between molecules and structures including the subunits that form the fibers in (1) and to assess the effects of physiologic and other interventions on such interactions; (5) surface topography of HSP104 particles and of Sup35 when exposed to Hsp104, (6) follow in time structural changes in Sup35 when Hsp104 is sufficiently overexpressed so as to """"""""cure"""""""" cells of the [PSI+] element, and (7) physical characteristics of the proteins.
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