Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California 92037, United States.
Department Chemistry and Biochemistry, University of California San Diego, La Jolla, California 92093, United States.
Biochemistry. 2023 Aug 1;62(15):2252-2256. doi: 10.1021/acs.biochem.3c00262. Epub 2023 Jul 17.
Elucidating the structure and interactions of proteins in native environments is a fundamental goal of structural biology. Nuclear magnetic resonance (NMR) spectroscopy is well suited for this task but often suffers from low sensitivity, especially in complex biological settings. Here, we use a sensitivity-enhancement technique called dynamic nuclear polarization (DNP) to overcome this challenge. We apply DNP to capture the membrane interactions of the outer membrane protein Ail, a key component of the host invasion pathway of . We show that the DNP-enhanced NMR spectra of Ail in native bacterial cell envelopes are well resolved and enriched in correlations that are invisible in conventional solid-state NMR experiments. Furthermore, we demonstrate the ability of DNP to capture elusive interactions between the protein and the surrounding lipopolysaccharide layer. Our results support a model where the extracellular loop arginine residues remodel the membrane environment, a process that is crucial for host invasion and pathogenesis.
阐明蛋白质在天然环境中的结构和相互作用是结构生物学的一个基本目标。核磁共振（NMR）光谱技术非常适合这项任务，但通常灵敏度较低，特别是在复杂的生物环境中。在这里，我们使用一种称为动态核极化（DNP）的灵敏度增强技术来克服这一挑战。我们应用 DNP 来捕捉外膜蛋白 Ail 的膜相互作用，Ail 是宿主入侵途径的关键组成部分。我们表明，在天然细菌细胞包膜中，AIL 的 DNP 增强 NMR 光谱具有良好的分辨率，并富集了在常规固态 NMR 实验中不可见的相关性。此外，我们证明了 DNP 能够捕捉到蛋白质与周围脂多糖层之间难以捉摸的相互作用。我们的结果支持一种模型，即细胞外环精氨酸残基重塑膜环境，这一过程对于宿主入侵和发病机制至关重要。
