Beriashvili David, Yao Ru, D'Amico Francesca, Krafčíková Michaela, Gurinov Andrei, Safeer Adil, Cai Xinyi, Mulder Monique P C, Liu Yangping, Folkers Gert E, Baldus Marc
NMR Spectroscopy, Bijvoet Center for Biomolecular Research, Utrecht University Padualaan 8 3584 CH Utrecht The Netherlands
Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnostics, School of Pharmacy, Tianjin Medical University Tianjin 300070 P. R. China.
Chem Sci. 2023 Sep 5;14(36):9892-9899. doi: 10.1039/d3sc02117c. eCollection 2023 Sep 20.
Studying the structural aspects of proteins within sub-cellular compartments is of growing interest. Dynamic nuclear polarization supported solid-state NMR (DNP-ssNMR) is uniquely suited to provide such information, but critically lacks the desired sensitivity and resolution. Here we utilize SNAPol-1, a novel biradical, to conduct DNP-ssNMR at high-magnetic fields (800 MHz/527 GHz) inside HeLa cells and isolated cell nuclei electroporated with [C,N] labeled ubiquitin. We report that SNAPol-1 passively diffuses and homogenously distributes within whole cells and cell nuclei providing ubiquitin spectra of high sensitivity and remarkably improved spectral resolution. For cell nuclei, physical enrichment facilitates a further 4-fold decrease in measurement time and provides an exclusive structural view of the nuclear ubiquitin pool. Taken together, these advancements enable atomic interrogation of protein conformational plasticity at atomic resolution and with sub-cellular specificity.
研究亚细胞区室中蛋白质的结构方面越来越受到关注。动态核极化支持的固态核磁共振（DNP-ssNMR）特别适合提供此类信息，但严重缺乏所需的灵敏度和分辨率。在这里，我们利用一种新型双自由基SNAPol-1，在HeLa细胞内以及用[C,N]标记的泛素进行电穿孔的分离细胞核中，在高磁场（800 MHz/527 GHz）下进行DNP-ssNMR。我们报告称，SNAPol-1在整个细胞和细胞核内被动扩散并均匀分布，提供了高灵敏度和显著提高的光谱分辨率的泛素光谱。对于细胞核，物理富集有助于将测量时间进一步缩短4倍，并提供核泛素池的独特结构视图。综上所述，这些进展使得能够在原子分辨率下并具有亚细胞特异性地对蛋白质构象可塑性进行原子级探究。
