Abstract
Magic angle spinning (MAS) solid-state nuclear magnetic resonance (NMR) is a powerful method for structure determination of insoluble biomolecules. However, structure determination by MAS solid-state NMR remains challenging because it is difficult to obtain a sufficient amount of distance restraints owing to spectral complexity. Collection of distance restraints from paramagnetic relaxation enhancement (PRE) is a promising approach to alleviate this barrier. However, the precision of distance restraints provided by PRE is limited in solid-state NMR because of incomplete averaged interactions and intermolecular PREs. In this report, the backbone structure of the B1 domain of streptococcal protein G (GB1) has been successfully determined by combining the CS-Rosetta protocol and qualitative PRE restraints. The derived structure has a Cα RMSD of 1.49 Å relative to the X-ray structure. It is noteworthy that our protocol can determine the correct structure from only three cysteine-EDTA-Mn2+ mutants because this number of PRE sites is insufficient when using a conventional structure calculation method based on restrained molecular dynamics and simulated annealing. This study shows that qualitative PRE restraints can be employed effectively for protein structure determination from a limited conformational sampling space using a protein fragment library.
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Acknowledgments
This work was supported by a Grant-in-Aid for JSPS Fellows (No. 26-4060), and a Grant-in-Aid for Scientific Research on Innovative Areas (No. 26120501) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. This work was performed under the Collaborative Research Program of the Institute for Protein Research, Osaka University.
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Tamaki, H., Egawa, A., Kido, K. et al. Structure determination of uniformly 13C, 15N labeled protein using qualitative distance restraints from MAS solid-state 13C-NMR observed paramagnetic relaxation enhancement. J Biomol NMR 64, 87–101 (2016). https://doi.org/10.1007/s10858-015-0010-0
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DOI: https://doi.org/10.1007/s10858-015-0010-0