Structure determination of uniformly (13)C, (15)N labeled protein using qualitative distance restraints from MAS solid-state (13)C-NMR observed paramagnetic relaxation enhancement

• Published in: Journal of biomolecular NMR Vol. 64; no. 1; pp. 87 - 101
• Main Authors: Tamaki, Hajime, Egawa, Ayako, Kido, Kouki, Kameda, Tomoshi, Kamiya, Masakatsu, Kikukawa, Takashi, Aizawa, Tomoyasu, Fujiwara, Toshimichi, Demura, Makoto
• Format: Journal Article
• Language: English
• Published: Netherlands 01.01.2016
• Subjects: Cysteine; Metals - chemistry; Models, Molecular; Molecular Conformation; Mutation; Nuclear Magnetic Resonance, Biomolecular - methods; Proteins - chemistry; Proteins - genetics; Solid-state NMR; Paramagnetic relaxation enhancement; Magic-angle spinning; Protein structure; CS-Rosetta
• ISSN: 1573-5001
• DOI: 10.1007/s10858-015-0010-0

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-Mn(2+) 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.