Structural study of monomeric, oligomeric and fibrillar yeast prion protein Sup35

描述（由申请人提供）：朊病毒是一种蛋白质，可以转变为自我延续的感染性构象。朊病毒的复制能力，形成结构不同的菌株，并建立和克服物种之间的传播障碍知之甚少。使用表面结合肽阵列，发现初级序列的小元件控制朊病毒状态的切换，对酵母朊病毒Sup35具有精确的特异性。这些相同的序列元件支配不同的自我永存构象（朊病毒株）的形成，并决定来自两种不同酵母菌株的Sup35的种特异性接种活性。这些序列元件的氨基酸组成几乎相同，但它们不发生交叉反应。这些区域的结构差异是什么，导致了物种屏障和菌株特异性？ 朊病毒蛋白的结构研究通常由于以下事实而复杂化：在天然条件下，高浓度朊病毒蛋白样品将在实验的时间尺度上（或更快）自发地形成纤维。为了捕获NM的单体形式进行结构研究，我们将使用NM的突变体版本与受损的聚集动力学，样品中的热休克蛋白104和反胶束中结合节段性同位素标记和改进的NMR仪器的存在下，询问Sc和Ca NM的纤维成核区域的结构。选择性地仅识别成熟寡聚物种类的抗体可以使寡聚物的溶液不确定地保持稳定。为了分离NM的寡聚体形式，我们将使用用于Sc和Ca NM的酵母表面展示产生各种单链可变片段（scFv）。对应于这些蛋白质的鉴定序列元件的肽的结构将使用溶液NMR方法在与未标记的scFv的复合物中解析。为了分离NM纤维的有趣的结构元素，我们将尝试阻断或消除部分形成的纤维的聚合界面。淀粉样蛋白聚集体越来越多地参与人类疾病，包括朊病毒性脑病、非感染性神经退行性疾病和系统性淀粉样变性。可溶性朊病毒蛋白通过一种被认为是有毒物质的途径上的寡聚中间体形成淀粉样纤维。深入了解Sup35NM的分子结构，从两个不同种类的酵母在单体，寡聚体和纤维将有助于解释如何一种蛋白质可以在朊病毒和非朊病毒状态之间切换，并提供一个结构合理的朊病毒感染的物种屏障。 朊病毒蛋白质的详细结构将有助于为针对淀粉样疾病的药物设计提供信息。

DESCRIPTION (provided by applicant): Prions are proteins that can switch to self-perpetuating, infectious conformations. The abilities of prions to replicate, form structurally distinct strains, and to establish and overcome transmission barriers between species are poorly understood. Using arrays of surface-bound peptides, small elements of primary sequence were found to control the switch to the prion state with exquisite specificity for the yeast prion Sup35. These same sequence elements govern the formation of distinct self-perpetuating conformations (prion strains) and determine species specific seeding activities for Sup35 from two different yeast strains. The amino acid compositon of these sequences elements is almost identical, but they do not cross-react. What are the structural differences in these regions that account for the species barrier and strain specificity? Structural investigation of prion proteins is often complicated by the fact that under native conditions, high concentration prion protein samples will spontaneously form fibers on (or faster than) the timescale of the experiment. To trap the monomeric form of NM for structural study, we will use mutant versions of NM with impaired aggregation kinetics, samples made in the presence of Hsp104 and encapusulation in reverse micelles in combination with segmental isotopic labeling and improved NMR instrumentation to interrogate the structure of the fiber nucleating regions of Sc and Ca NM. Antibodies that selectively recognize only the mature oligomeric species can keep solutions of oligomers stable indefinately. To isolate the oligomeric form of NM, we will raise a variety of single chain variable fragments (scFv) using yeast surface display for Sc and Ca NM. The structure of the peptides corresponding to the identified sequence elements of these proteins will be solved in complex with the unlabled scFv using solution NMR methods. To isolate the interesting structural elements of NM fibers, we will try to block or eliminate the polymerization interface of partially formed fibers. Amyloid protein aggregates have been increasingly implicated in human diseases, including prion-based encephalopathies, noninfectious neurodegenerative diseases and systemic amyloidoses. Soluble prion proteins form amyloid fibers via an on-pathway oligomeric intermediate that is thought to be the toxic species. Insight into the molecular structure of Sup35NM from two different species of yeast in the monomeric, oligomeric and fibers will help explain how one protein can switch between the prion and non-prion states and provide a structural rational for the species barrier for prion infection. Detailed structures of prion proteins will help inform drug design efforts against amyloid diseases.