Abstract
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 [ 13C, 15N] 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.
Original language | English |
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Pages (from-to) | 9892-9899 |
Number of pages | 8 |
Journal | Chemical Science |
Volume | 14 |
Issue number | 36 |
DOIs | |
Publication status | Published - 5 Sept 2023 |
Bibliographical note
Publisher Copyright:© 2023 The Royal Society of Chemistry.
Funding
We thank Johan van der Zwan for technical support, Prof. Dr A. J. J. M. Bonvin for helpful discussions, and Dr Ilya Grigoriev of the Biology Imaging Center (BIC), Department of Biology, Utrecht University for confocal microscopy access and training. This work was supported by the Dutch Science Foundation NWO (grants 175.010.2009.002, 718.015.001 and 711.018.008 to M. B.) and by uNMR-NL-grid, a distributed state-of-the-art Magnetic Resonance Facility for the Netherlands (grant 184.035.002). In addition, our work was funded in part by iNEXT-Discovery (grant number 871037), a Horizon 2020 program of the European Commission, and the National Natural Science Foundation of China (No. 21871210, 22174099, and 21572161 to Y. P. L). F. D. acknowledges support from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no. 813599. And M. P. C. M. acknowledges support from NWO (VIDI Grant VI. 213.110). The graphical abstract was prepared with Bio-render software.
Funders | Funder number |
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Dutch Science Foundation NWO | 184.035.002, 711.018.008, 175.010.2009.002, 718.015.001, 871037 |
Horizon 2020 Framework Programme | |
H2020 Marie Skłodowska-Curie Actions | 813599 |
European Commission | |
National Natural Science Foundation of China | 21572161, 21871210, 22174099 |
Nederlandse Organisatie voor Wetenschappelijk Onderzoek | 213.110 |
Horizon 2020 |