Photo-induced cross-linking of unmodified proteins (PICUP) applied to amyloidogenic peptides.
Photo-induced cross-linking of unmodified proteins (PICUP) applied to amyloidogenic peptides.
复制标题
DOI：
10.3791/1071
复制
发表时间：
2009-01-12
期刊：
影响因子：
--
通讯作者：
Bitan, Gal
中科院分区：
文献类型：
--
作者：
Rahimi, Farid;Maiti, Panchanan;Bitan, Gal
淀粉样蛋白质形成毒性寡聚体的组装是蛋白质错误折叠疾病（包括阿尔茨海默病、帕金森病和亨廷顿病、遗传性肌萎缩侧索硬化症和2型糖尿病）发病机理中的开创性事件。由于这些蛋白质组装体的亚稳态性质，很难使用经典方法（例如电泳、色谱、荧光或动态光散射）定量评估它们的寡聚体尺寸分布。淀粉样蛋白的低聚物以亚稳态混合物的形式存在，其中低聚物解离成单体并同时缔合成更大的组装体。PICUP通过共价交联稳定低聚物群体，并且当与分级分离方法如十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）或尺寸排阻色谱（SEC）结合时，PICUP提供交联前存在的低聚物尺寸分布的快照。因此，PICUP能够可视化和定量分析亚稳态蛋白质群体，并可用于监测序列修饰和寡聚化之间的组装和破译关系（1）。从机理上讲，PICUP涉及在电子受体存在下，通过可见光照射将三（联吡啶）Ru（II）络合物（RuBpy）中的Ru（2+）光氧化为Ru（3+）。Ru（3+）是一种强的单电子氧化剂，能够从邻近的蛋白质分子中夺取一个电子，产生蛋白质自由基（1，2）。自由基是不稳定的，高活性的物种，因此通过各种分子内和分子间反应迅速消失。自由基可以利用未配对电子的高能量与另一个蛋白质单体反应形成二聚体自由基，其随后失去氢原子并形成稳定的共价连接的二聚体。然后，二聚体可以通过类似的机理与单体或其他二聚体进一步反应以形成更高级的低聚物。PICUP相对于其它光或化学交联方法的优点（3，4）包括短（
The assembly of amyloidogenic proteins into toxic oligomers is a seminal event in the pathogenesis of protein misfolding diseases, including Alzheimer's, Parkinson's, and Huntington's diseases, hereditary amyotrophic lateral sclerosis, and type 2 diabetes. Owing to the metastable nature of these protein assemblies, it is difficult to assess their oligomer size distribution quantitatively using classical methods, such as electrophoresis, chromatography, fluorescence, or dynamic light scattering. Oligomers of amyloidogenic proteins exist as metastable mixtures, in which the oligomers dissociate into monomers and associate into larger assemblies simultaneously. PICUP stabilizes oligomer populations by covalent cross-linking and when combined with fractionation methods, such as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or size-exclusion chromatography (SEC), PICUP provides snapshots of the oligomer size distributions that existed before cross-linking. Hence, PICUP enables visualization and quantitative analysis of metastable protein populations and can be used to monitor assembly and decipher relationships between sequence modifications and oligomerization(1). Mechanistically, PICUP involves photo-oxidation of Ru(2+) in a tris(bipyridyl)Ru(II) complex (RuBpy) to Ru(3+) by irradiation with visible light in the presence of an electron acceptor. Ru(3+) is a strong one-electron oxidizer capable of abstracting an electron from a neighboring protein molecule, generating a protein radical(1,2). Radicals are unstable, highly-reactive species and therefore disappear rapidly through a variety of intra- and intermolecular reactions. A radical may utilize the high energy of an unpaired electron to react with another protein monomer forming a dimeric radical, which subsequently loses a hydrogen atom and forms a stable, covalently-linked dimer. The dimer may then react further through a similar mechanism with monomers or other dimers to form higher-order oligomers. Advantages of PICUP relative to other photo- or chemical cross-linking methods(3,4) include short (