Probing the Conformation of a Prion Protein Fibril with Hydrogen Exchange
Probing the Conformation of a Prion Protein Fibril with Hydrogen Exchange
复制标题
DOI：
10.1074/jbc.m110.114504
复制
发表时间：
2010-10-15
影响因子：
4.8
通讯作者：
Wemmer, David E.
中科院分区：
文献类型：
--
作者：
Damo, Steven M.;Phillips, Aaron H.;Wemmer, David E.
朊病毒蛋白PrP（89-143，P101 L）的一个片段，带有与家族性朊病毒病有关的突变，当脑内注射到表达含有P101 L突变的全长PrP的转基因小鼠中时，形成的原纤维已显示出诱导朊病毒病。在这项研究中，我们利用酰胺氢交换测量探测其纤维状形式的肽的组织。我们首先通过串联蛋白水解、液相色谱和质谱（HXMS），然后通过交换淬灭NMR确定氢交换的程度。虽然单酰胺分辨率提供了NMR测量，HXMS是非常适合于天然朊病毒的研究，因为它不需要标记与NMR活性同位素。因此，从感染动物中获得的天然朊病毒可以与本文研究的模型系统如PrP（89-143，P101 L）进行比较。在我们的研究中，我们发现了两个序列片段，即残基102-109和117-136，具有高水平的交换保护作用。此外，有一个区域，显示交换行为与构象异构转向的存在相一致。我们讨论了我们的数据方面提出的几个结构模型的传染性朊蛋白聚集体和突出HXMS作为少数几个技术非常适合研究天然朊病毒。
A fragment of the prion protein, PrP(89-143, P101L), bearing a mutation implicated in familial prion disease, forms fibrils that have been shown to induce prion disease when injected intracerebrally into transgenic mice expressing full-length PrP containing the P101L mutation. In this study, we utilize amide hydrogen exchange measurements to probe the organization of the peptide in its fibrillar form. We determined the extent of hydrogen exchange first by tandem proteolysis, liquid chromatography, and mass spectrometry (HXMS) and then by exchange-quenched NMR. Although single amide resolution is afforded by NMR measurements, HXMS is well suited to the study of natural prions because it does not require labeling with NMR active isotopes. Thus, natural prions obtained from infected animals can be compared with model systems such as PrP(89-143, P101L) studied here. In our study, we find two segments of sequence that display a high level of protection from exchange, residues 102-109 and 117-136. In addition, there is a region that displays exchange behavior consistent with the presence of a conformationally heterogeneous turn. We discuss our data with respect to several structural models proposed for infectious PrP aggregates and highlight HXMS as one of the few techniques well suited to studying natural prions.