DNP enhanced MAS solid-state NMR of biological systems in their native context

我们建议使用DNP（动态核极化）MAS固态核磁共振来确定膜蛋白和淀粉样蛋白聚集体在其原生细胞环境中的结构，而无需纯化和重构。该方法将应用于膜蛋白，我们表征以前使用标准核磁共振方法。我们将特别关注膜蛋白Mistic（一种膜蛋白伴侣）和EmrE（一种多药耐药转运蛋白）。此外，我们将研究阿尔茨海默病ß-淀粉样肽，并在神经元细胞的背景下表征其结构。DNP在所有这些项目中都是必不可少的，因为可以将大量的同位素富集物质装入MAS转子中，并且其浓度通常太少，无法使用标准溶液状态或固态核磁共振波谱方法进行适当的结构表征。

We propose to use DNP (Dynamic Nuclear Polarization) MAS solid-state NMR to determine the structure of membrane proteins and amyloid aggregates in their native cellular environment without the need for purification and reconstitution. The method will be applied to membrane proteins that we characterized previously employing standard NMR methodologies. In particular, we will focus on the membrane protein Mistic, a membrane protein chaperone, and EmrE, a multidrug resistance transporter. Furthermore, we will investigate the Alzheimer’s disease ß-amyloid peptide and characterize its structure in the context of neuronal cells. DNP is essential in all these projects as the available amount of isotopically enriched material, which can be packed into a MAS rotor, and its concentration is typically too little to enable a proper structural characterization using standard solution-state or solid-state NMR spectroscopy methods.