Abstract
Elucidating at atomic level how proteins interact and are chemically modified in cells represents a leading frontier in structural biology. We have developed a tailored solid-state NMR spectroscopic approach that allows studying protein structure inside human cells at atomic level under high-sensitivity dynamic nuclear polarization (DNP) conditions. We demonstrate the method using ubiquitin (Ub), which is critically involved in cellular functioning. Our results pave the way for structural studies of larger proteins or protein complexes inside human cells, which have remained elusive to in-cell solution-state NMR spectroscopy due to molecular size limitations.
| Original language | English |
|---|---|
| Pages (from-to) | 12969-12973 |
| Number of pages | 5 |
| Journal | Angewandte Chemie - International Edition |
| Volume | 58 |
| Issue number | 37 |
| DOIs | |
| Publication status | Published - 9 Sept 2019 |
Funding
We are indebted to Professor Paul Tordo and Dr. Olivier Ouari (Aix-Marseille Université, Marseille) for providing AMUpol and PyPol-MTSSL. We thank Mark Daniëls for technical support as well as helpful discussions, along with Suzan Leemans for the latter. We thank Hugo van Ingen and Rolf Boelens for providing access to the 900 MHz solution-state NMR instrument. We are grateful to Dr. Philipp Selenko and his group (Weizmann Institute) for sharing their expertise with protein delivery into eukaryotic cells using electropora-tion. This work was funded in part by the Netherlands Organisation for Scientific Research (NWO) (Graduate Program; project 022.005.029, Netherlands≫ Magnetic Reso- nance Research School, NMARRS, a VICI grant to H.O. as well as grants 700.26.121 and 700.10.443 to M.B.) and iNEXT (project number 653706), a Horizon 2020 program of the European Union.
Keywords
- dynamic nuclear polarization
- in-cell NMR
- protein–protein interactions
- solid-state NMR
- ubiquitination