YEAST PRION DYNAMICS AND CHAPERONE INTERACTIONS

蛋白质构象和组装的变化支配着细胞生物学的大多数过程。原子或扫描力显微镜（AFM）是一种新的成像方式，具有强大而独特的能力，可以直接研究这些过程，因为它具有高空间分辨率（坚硬表面上的原子），扩展的力范围（高达10-15N）足以测量几乎任何分子相互作用，纳米级控制，功能化能力，因此它可以作为特定生物分子的非常特定的传感器，在操作和制备中相对缺乏破坏性。以及在干燥和生理溶液中成像标本的能力。这些功能将AFM与传统的成像模式区分开来，并为结构生物学开辟了新的途径。这项提议汇集了苏珊·林德奎斯特实验室的专业知识，该实验室在研究细胞质遗传遗传因素方面处于领先地位，而阿恩斯多夫实验室一直在为生物医学研究开发AFM。最有趣的细胞质遗传基因元素是传染性监狱蛋白，它导致传染性海绵状脑病，如牛的“疯牛病”和人类的各种逐渐致命的神经退行性疾病。朊病毒蛋白通常存在于细胞膜中，最近的研究支持这一假设，即该蛋白的构象异常形式是导致这些疾病的感染性颗粒。酵母中两个细胞质遗传的遗传元件通过类似的机制繁殖，在翻译保真度或氮代谢方面产生可遗传的变化。[PSI+]是最具特征的酵母朊病毒，被认为是在核编码的Sup35蛋白（翻译终止装置的一个亚基）中自我延续的构象改变。[PSI+]元件的繁殖取决于作为伴侣的热休克蛋白104 （HSP104）的特定浓度，但有点矛盾的是，HSP104的过表达可以“治愈”[PSI+]元件的细胞并恢复正常的翻译保真度。我们将用原子力显微镜研究：(1)Sup35的表面结构，其组成亚基“构建块”，可能的中间体和聚集体；（二）第（一）项的时间进程；(3)自续的亚基聚合模式及其与[PSI+]应变变化的关系；(4)分子和结构（包括在(1)中形成纤维的亚基）之间相互作用的力，并评估生理和其他干预对这种相互作用的影响；(5) HSP104颗粒和Sup35暴露于HSP104时的表面形貌；(6)当HSP104充分过表达以“治愈”[PSI+]元素的细胞时，Sup35的时间结构变化；(7)蛋白质的物理特性。

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.