Nano-mole scale side-chain signal assignment by1H-detected protein solid-state NMR by ultra-fast magic-angle spinning and stereo-array isotope labeling

Songlin Wang, Sudhakar Parthasarathy, Yusuke Nishiyama, Yuki Endo, Takahiro Nemoto, Kazuo Yamauchi, Tetsuo Asakura, Mitsuhiro Takeda, Tsutomu Terauchi, Masatsune Kainosho, Yoshitaka Ishii

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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.

Original languageEnglish
Article numbere0122714
JournalPLoS One
Volume10
Issue number4
DOIs
Publication statusPublished - Apr 9 2015
Externally publishedYes

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ASJC Scopus subject areas

  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

Cite this

Wang, S., Parthasarathy, S., Nishiyama, Y., Endo, Y., Nemoto, T., Yamauchi, K., Asakura, T., Takeda, M., Terauchi, T., Kainosho, M., & Ishii, Y. (2015). Nano-mole scale side-chain signal assignment by1H-detected protein solid-state NMR by ultra-fast magic-angle spinning and stereo-array isotope labeling. PLoS One, 10(4), [e0122714]. https://doi.org/10.1371/journal.pone.0122714