title: 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.
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-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.
authors.affiliation: Graduate School of Life Science, Hokkaido University, Sapporo, Japan.
authors.forename: Hajime
authors.initials: H
authors.lastname: Tamaki
authors.affiliation: Institute for Protein Research, Osaka University, Osaka, Japan.
authors.forename: Ayako
authors.initials: A
authors.lastname: Egawa
authors.affiliation: Graduate School of Life Science, Hokkaido University, Sapporo, Japan.
authors.forename: Kouki
authors.initials: K
authors.lastname: Kido
authors.affiliation: Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology, Tokyo, Japan.
authors.forename: Tomoshi
authors.initials: T
authors.lastname: Kameda
authors.affiliation: Faculty of Advanced Life Science, Hokkaido University, Sapporo, Japan.
authors.forename: Masakatsu
authors.initials: M
authors.lastname: Kamiya
authors.affiliation: Faculty of Advanced Life Science, Hokkaido University, Sapporo, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Kikukawa
authors.affiliation: Faculty of Advanced Life Science, Hokkaido University, Sapporo, Japan.
authors.forename: Tomoyasu
authors.initials: T
authors.lastname: Aizawa
authors.affiliation: Institute for Protein Research, Osaka University, Osaka, Japan.
authors.forename: Toshimichi
authors.initials: T
authors.lastname: Fujiwara
authors.affiliation: Faculty of Advanced Life Science, Hokkaido University, Sapporo, Japan. demura@sci.hokudai.ac.jp.
authors.forename: Makoto
authors.initials: M
authors.lastname: Demura
mesh_terms: D003545:Cysteine; D008670:Metals / Q000737:chemistry; D008958:Models, Molecular; D008968:Molecular Conformation; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011506:Proteins / Q000737:chemistry* / Q000235:genetics
chemical_list: D008670:Metals; D011506:Proteins; D003545:Cysteine
keywords: CS-Rosetta; Magic-angle spinning; Paramagnetic relaxation enhancement; Protein structure; Solid-state NMR