Nano-Mole Scale Side-Chain Signal Assignment by 1H-Detected Protein Solid-State NMR by Ultra-Fast Magic-Angle Spinning and Stereo-Array Isotope Labeling
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Date
2015-04
Authors
Wang, Songlin
Parthasarathy, Sudhakar
Nishiyama, Yusuke
Endo, Yuki
Nemoto, Takahiro
Yamauchi, Kazuo
Asakura, Tetsuo
Takeda, Mitsuhiro
Terauchi, Tsutomu
Kainosho, Masatsune
Journal Title
Journal ISSN
Volume Title
Publisher
Public Library of Science
Abstract
We present a general approach in 1H-detected 13C solid-state NMR (SSNMR) for side-chain signal assignments of 10-50 nmol quantities of proteins using a combination of a high magnetic field, ultra-fast magic-angle spinning (MAS) at ~80 kHz, and stereo-array-isotope-labeled (SAIL) proteins [Kainosho M. et al., Nature 440, 52–57, 2006]. First, we demonstrate that 1H indirect detection improves the sensitivity and resolution of 13C SSNMR of SAIL proteins for side-chain assignments in the ultra-fast MAS condition. 1H-detected SSNMR was performed for micro-crystalline ubiquitin (~55 nmol or ~0.5mg) that was SAIL-labeled at seven isoleucine (Ile) residues. Sensitivity was dramatically improved by 1H-detected 2D 1H/13C SSNMR by factors of 5.4-9.7 and 2.1-5.0, respectively, over 13C-detected 2D 1H/13C SSNMR and 1D 13C CPMAS, demonstrating that 2D 1H-detected SSNMR offers not only additional resolution but also sensitivity advantage over 1D 13C detection for the first time. High 1H resolution for the SAIL-labeled side-chain residues offered reasonable resolution even in the 2D data. A 1H-detected 3D 13C/13C/1H experiment on SAIL-ubiquitin provided nearly complete 1H and 13C assignments for seven Ile residues only within ~2.5 h. The results demonstrate the feasibility of side-chain signal assignment in this approach for as little as 10 nmol of a protein sample within ~3 days. The approach is likely applicable to a variety of proteins of biological interest without any requirements of highly efficient protein expression systems.
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Keywords
Research Subject Categories::NATURAL SCIENCES::Chemistry, nano-mole, magic-angle spinning, MAS, stereo-array-isotope-labeled, SAIL, protein expression system
Citation
: Wang S, Parthasarathy S, Nishiyama Y, Endo Y, Nemoto T, Yamauchi K, et al. (2015) NanoMole Scale Side-Chain Signal Assignment by 1 HDetected Protein Solid-State NMR by Ultra-Fast Magic-Angle Spinning and Stereo-Array Isotope Labeling. PLoS ONE 10(4): e0122714. doi:10.1371/ journal.pone.0122714