Structural Studies of Prion Fibrils and Other Protein Fibrils

朊病毒原纤维和其他蛋白质原纤维的结构研究

• 批准号: 7967835
• 负责人: ROBERT TYCKO
• 金额: $ 13.05万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7967835
• 关键词: Adhesives; Adopted; Amino Acid Sequence; Amyloid; Amyloid Fibrils; Amyloid beta-Protein; Area; Bacteria; Biological Process; Disease; Electron Microscope; Electron Microscopy; Goals; Hydration status; Image; Investigation; Knowledge; Length; Light; Literature; Mammals; Melanins; Methods; Molecular; Molecular Structure; Organism; Phenotype; Prions; Production; Protein Structure Initiative; Proteins; RNA Interference; Role; Structure; Suggestion; Surface; System; Yeasts; amyloid fibril formation; beta pleated sheet; instrumentation; intermolecular interaction; melanocyte; mutant; solid state nuclear magnetic resonance; sup35; transmission process; yeast prion

Progress in FY2009 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. INITIAL 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 Pmel (which catalyzes melanin production in mammalian melanocytes). Neither CsgA nor Pmel 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. Pmel fibrils give very high-quality solid state NMR spectra, indicating that full molecular structure determination for Pmel fibrils may be possible in the next fiscal year. 3. METHOD FOR MASS-PER-LENGTH DETERMINATION IN AMYLOID AND PRION FIBRILS. We demonstrated that the mass-per-length (kDa/nm) of a fibril can be determined quantitatively from dark field electron microscope images obtained with the tilted-beam mode of a conventional transmission electron microscope. This makes mass-per-length determination (an essential component of molecular structure determination) possible with readily-available instrumentation. We are now applying the new method in studies of functional amyloids, prion fibrils, and related systems.

2009财年在以下领域取得了进展： 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初步研究。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PMEL(它催化哺乳动物黑素细胞中黑色素的产生)。CsgA和PMEL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的β-折叠结构。相反，它们可能具有β螺旋结构。PMEL纤维提供了非常高质量的固体核磁共振谱，这表明在下一财年可能有可能确定PMEL纤维的全分子结构。 3.淀粉样蛋白和蛋白原纤维单位长度质量的测定方法。我们证明了利用常规电子显微镜的倾斜光束模式获得的暗场电子显微镜图像可以定量地确定纤维的每长度质量(kDa/nm)。这使得每长度质量的测定(分子结构测定的一个重要组成部分)可以通过现成的仪器来实现。我们现在正在将这种新方法应用于功能淀粉样蛋白、蛋白原纤维和相关系统的研究。