A new carbamidemethyl-linked lanthanoid chelating tag for PCS NMR spectroscopy of proteins in living HeLa cells
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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.
KeywordsIn-cell NMR Cultured human cells Paramagnetic NMR Lanthanoid-chelating tag Electroporation
The authors thank Dr Jin Inoue and Ms Maho Ishikawa for the preparation of ubiquitin mutant proteins, Mr Junya Iinuma, Ms Mayu Nishizawa, and Dr Daichi Morimoto for the experiments on the reactivity of M8-CAM-I to cysteine thiol groups, and Drs Hiromasa Yagi and Sundaresan Rajesh for a critical reading of the manuscript. Molecular graphics and analyses were performed with the UCSF Chimera package (http://www.rbvi.ucsf.edu/chimera). Chimera is developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (Pettersen et al. 2004) (supported by NIGMS P41-GM103311). This work was supported by the Funding Program for Next Generation World-Leading Researchers (NEXT), Grants-in-Aid for Challenging Exploratory Research (JSPS KAKENHI Grant Number JP15K14463 and JP15K14494) and Grant-in-Aid for Scientific Research C (JSPS KAKENHI Grant Number JP 15K06979) from Japan Society for the Promotion of Science (JSPS), Grants-in-Aid for Scientific Research on Innovative Areas (MEXT KAKENHI Grant Numbers JP25120003, JP26102538, JP15H01645, JP16H00779, JP16H00847) and the NMR Platform from the Japanese Ministry of Education, Sports, Culture, Science, and Technology (MEXT), and the Core Research for Evolutional Science and Technology (CREST) program from the Japan Science and Technology Agency (JST).