Journal
JOURNAL OF BIOMOLECULAR NMR
Volume 66, Issue 2, Pages 99-110Publisher
SPRINGER
DOI: 10.1007/s10858-016-0059-4
Keywords
In-cell NMR; Cultured human cells; Paramagnetic NMR; Lanthanoid-chelating tag; Electroporation
Categories
Funding
- NIGMS [P41-GM103311]
- Funding Program for Next Generation World-Leading Researchers (NEXT), (JSPS KAKENHI Grant) from Japan Society for the Promotion of Science (JSPS) [JP15K14463, JP15K14494, JP 15K06979]
- MEXT KAKENHI Grant [JP25120003, JP26102538, JP15H01645, JP16H00779, JP16H00847]
- NMR Platform from the Japanese Ministry of Education, Sports, Culture, Science, and Technology (MEXT)
- Core Research for Evolutional Science and Technology (CREST) program from the Japan Science and Technology Agency (JST)
- Grants-in-Aid for Scientific Research [15K12760, 16H00847, 15K14494, 16H00779, 15K06979, 16H01167, 15H01645] Funding Source: KAKEN
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Structural analyses of proteins under macromolecular crowding inside human cultured cells by in-cell NMR spectroscopy are crucial not only for explicit understanding of their cellular functions but also for applications in medical and pharmaceutical sciences. In-cell NMR experiments using human cultured cells however suffer from low sensitivity, thus pseudocontact shifts from protein-tagged paramagnetic lanthanoid ions, analysed using sensitive heteronuclear two-dimensional correlation NMR spectra, offer huge potential advantage in obtaining structural information over conventional NOE-based approaches. We synthesised a new lanthanoid-chelating tag (M8-CAM-I), in which the eight-fold, stereospecifically methylated DOTA (M8) scaffold was retained, while a stable carbamidemethyl (CAM) group was introduced as the functional group connecting to proteins. M8-CAM-I successfully fulfilled the requirements for in-cell NMR: high-affinity to lanthanoid, low cytotoxicity and the stability under reducing condition inside cells. Large PCSs for backbone N-H resonances observed for M8-CAM-tagged human ubiquitin mutant proteins, which were introduced into HeLa cells by electroporation, demonstrated that this approach readily provides the useful information enabling the determination of protein structures, relative orientations of domains and protein complexes within human cultured cells.
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