Structural Studies of Prion Fibrils and Other Protein Fibrils

2010财年在以下方面取得了进展： 1.酵母蛋白的β-折叠结构的测定我们证明了三种酵母蛋白在其蛋白活性的淀粉样原纤维状态下采用注册的平行的β-折叠结构。其中包括产生URE3表型的Ure2p蛋白，产生PSI+表型的Sup35蛋白，以及产生PIN+表型的Rnq1蛋白。我们证明了在Ure2p和Sup35的突变体中保持了寄存器中平行的β-折叠结构，这些突变体在其Prion结构域中扰乱了氨基酸序列，并且这种结构不依赖于水合作用。这些结果应该会平息文献中关于酵母蛋白具有类似β-螺旋的结构，而不是注册的平行β-折叠结构的说法。 2.功能性淀粉样蛋白的研究。我们对两种被认为在淀粉样蛋白状态下具有生物学功能的蛋白质进行了初步研究，即CsgA(它在细菌表面形成所谓的卷曲纤维，具有粘连功能)和PMel17(它催化哺乳动物黑素细胞中黑色素的产生)。CsgA和PMel17纤维似乎都没有在酵母蛋白纤维和β-淀粉样蛋白纤维中发现的平行β-折叠结构。相反，它们可能具有β螺旋结构。对PMel17纤维的广泛研究表明，这些纤维是高度多态的，不同的多态在组成纤维核心的伪重复序列的同一性上是不同的。 3.胰岛素原纤维结构。对人胰岛素形成的纤维的固态核磁共振测量表明，天然的α-螺旋转化为β-链(与M.Weiss合作)。固体核磁共振数据对可能的分子结构施加了限制，表明文献中关于胰岛素原纤维的模型(在没有详细实验数据的情况下开发)是不正确的。胰岛素原纤维的形成是一个严重的公共卫生问题，特别是在缺乏冷藏的地区，因为储存的胰岛素在高温下往往会转化为原纤维。 4.哺乳动物的蛋白原纤维。对重组哺乳动物PrP(全长，叙利亚仓鼠序列)形成的纤维的固体核磁共振测量表明，这些纤维含有平行的β-折叠，并且纤维核心主要由靠近C-末端的30个残基片段形成(与I.V.Baskakov合作)。虽然这些纤维不像普恩那样具有传染性，但它们可能在许多方面类似于传染性PrP聚集体。

Progress in FY2010 was in the following areas: 1. DETERMINATION OF BETA-SHEET STRUCTURES IN YEAST PRIONS. We showed that three yeast prion proteins adopt in-register parallel beta-sheet structures in their prion-active, amyloid fibril states. These include the Ure2p protein that produces the URE3 phenotype, the Sup35 protein that produces the PSI+ phenotype, and the Rnq1 protein that produces the PIN+ phenotype. We showed that the in-register parallel beta-sheet structure is maintained in mutants of Ure2p and Sup35 that have scrambled amino acid sequences in their prion domains, and that this structure is not dependent on hydration. These results should quell suggestions in the literature that yeast prions have beta-helix-like structures, rather than in-register parallel beta-sheet structures. 2. INVESTIGATIONS OF FUNCTIONAL AMYLOIDS. We carried out initial studies of two proteins that are believed to have biological functions in their amyloid state, namely CsgA (which forms so-called curli fibrils on the surface of bacteria, which have an adhesive function) and Pmel17 (which catalyzes melanin production in mammalian melanocytes). Neither CsgA nor Pmel17 fibrils appear to have the in-register parallel beta-sheet structures found in yeast prion fibrils and in beta-amyloid fibrils. Instead, they may have beta-helical structures. An extensive study of Pmel17 fibrils shows that these fibrils are highly polymorphic, and that different polymorphs differ in the identity of pseudo-repeat sequences that comprise the fibril core. 3. INSULIN FIBRIL STRUCTURES. Solid state NMR measurements on fibrils formed by human insulin indicate conversion of native alpha-helices to beta-strands (collaboration with M. Weiss). The solid state NMR data place constraints on possible molecular structures, showing that models for insulin fibrils in the literature (developed in absence of detailed experimental data) are not correct. Insulin fibril formation is a serious public health problem, particularly in areas that lack refrigeration, as stored insulin tends to convert to fibrils at elevated temperatures. 4. MAMMALIAN PRION PROTEIN FIBRILS. Solid state NMR measurements on fibrils formed by recombinant mammalian PrP (full-length, Syrian hamster sequence) show that these fibrils contain parallel beta-sheets, and that the fibril core is formed primarily by a 30-residue segment near the C-terminus (collaboration with I.V. Baskakov). Although these fibrils are not infectious as prions, they may resemble infectious PrP aggregates in many respects.