Characterization of β-sheet structure in Ure2p1-89 yeast prion fibrils by solid-state nuclear magnetic resonance
Characterization of β-sheet structure in Ure2p1-89 yeast prion fibrils by solid-state nuclear magnetic resonance
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DOI：
10.1021/bi700826b
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发表时间：
2007-11-13
期刊：
影响因子：
2.9
通讯作者：
Tycko, Robert
中科院分区：
文献类型：
--
作者：
Baxa, Ulrich;Wickner, Reed B.;Tycko, Robert
残基1 - 89构成Ure2p蛋白的富含Asn和Gln的区段，并通过形成细胞内Ure2p淀粉样蛋白的核心产生酿酒酵母的[URE3]朊病毒。我们报告了固态核磁共振（NMR）测量的结果，探测由Ure2p（1 - 89）形成的淀粉样蛋白原纤维的分子结构，在体外。数据包括在特定位点被C-13标记的样品中的分子间磁偶极-偶极耦合的测量，以及均匀地被N-15和C-13标记的样品的二维N-15-C-13和C-13-C-13 NMR光谱。分子间偶极-偶极耦合表明，Ure2p（1 - 89）纤维中的β-折叠具有配准平行结构。配准平行P-片层结构允许Gln和Asn残基的侧链之间的极性拉链相互作用，并解释了[URE3]对残基1 - 89中的序列扰乱的耐受性。均匀标记的Ure2p（1 - 89）原纤维的二维NMR谱，即使在完全水合时，也显示NMR线宽超过由鹅柄孢霉的HET-s朊病毒蛋白的残基218 - 289形成的原纤维的固态NMR谱中的那些[如最初在Siemer，A. B.，Ritter，C.，恩斯特，M.，里克，R.，Meier，B. H.（2005）Angew.化学成分：Int.Ed.44，2441 - 2444中，并通过本文报道的测量证实]，其系数为3或更多，表明在Ure2p 1 - 89原纤维中在分子水平上的结构有序度较低。因此，固态NMR数据表明HET-s原纤维中非常高的结构有序度不是朊病毒蛋白的普遍特征，并且可能是HET-s在异核体不相容性中进化的生物学功能的结果。均匀标记的Ure2p（1 - 89）原纤维的二维NMR光谱中交叉峰强度的分析表明，氨基酸序列的某些部分可能不参与刚性P-片层结构，可能包括残基44和76之间的富含Asn的片段。
Residues 1-89 constitute the Asn- and Gln-rich segment of the Ure2p protein and produce the [URE3] prion of Saccharomyces cerevisiae by forming the core of intracellular Ure2p amyloid. We report the results of solid-state nuclear magnetic resonance (NMR) measurements that probe the molecular structure of amyloid fibrils formed by Ure2p(1-89), in vitro. Data include measurements of intermolecular magnetic dipole-dipole couplings in samples that are C-13-labeled at specific sites and two-dimensional N-15-C-13 and C-13-C-13 NMR spectra of samples that are uniformly N-15- and C-13-labeled. Intermolecular dipole-dipole couplings indicate that the beta-sheets in Ure2p(1-89) fibrils have an in-register parallel structure. An in-register parallel P-sheet structure permits polar zipper interactions among side chains of Gln and Asn residues and explains the tolerance of [URE3] to scrambling of the sequence in residues 1-89. Two-dimensional NMR spectra of uniformly labeled Ure2p(1-89) fibrils, even when fully hydrated, show NMR linewidths that exceed those in solid-state NMR spectra of fibrils formed by residues 218-289 of the HET-s prion protein of Podospora anserina [as originally reported in Siemer, A. B., Ritter, C., Ernst, M., Rick, R., and Meier, B. H. (2005) Angew. Chem., Int. Ed. 44, 2441-2444 and confirmed by measurements reported here] by factors of three or more, indicating a lower degree of structural order at the molecular level in Ure2p 1 -89 fibrils. The very high degree of structural order in HET-s fibrils indicated by solid-state NMR data is therefore not a universal characteristic of prion proteins, and is likely to be a consequence of the evolved biological function of HET-s in heterokaryon incompatibility. Analysis of cross peak intensities in two-dimensional NMR spectra of uniformly labeled Ure2p(1-89) fibrils suggests that certain portions of the amino acid sequence may not participate in a rigid P-sheet structure, possibly including portions of the Asn-rich segment between residues 44 and 76.