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

Research output: Contribution to journalArticle

10 Citations (Scopus)

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

Fingerprint

Isotope Labeling
isotope labeling
Magic angle spinning
spinning
Isotopes
Labeling
Nuclear magnetic resonance
isotopes
Proteins
proteins
Isoleucine
isoleucine
ubiquitin
Ubiquitin
Magnetic Fields
magnetic fields
protein synthesis
Magnetic fields
Crystalline materials

ASJC Scopus subject areas

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

Cite this

Nano-mole scale side-chain signal assignment by1H-detected protein solid-state NMR by ultra-fast magic-angle spinning and stereo-array isotope labeling. / Wang, Songlin; Parthasarathy, Sudhakar; Nishiyama, Yusuke; Endo, Yuki; Nemoto, Takahiro; Yamauchi, Kazuo; Asakura, Tetsuo; Takeda, Mitsuhiro; Terauchi, Tsutomu; Kainosho, Masatsune; Ishii, Yoshitaka.

In: PLoS One, Vol. 10, No. 4, e0122714, 09.04.2015.

Research output: Contribution to journalArticle

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, vol. 10, no. 4, e0122714. https://doi.org/10.1371/journal.pone.0122714
Wang, Songlin ; Parthasarathy, Sudhakar ; Nishiyama, Yusuke ; Endo, Yuki ; Nemoto, Takahiro ; Yamauchi, Kazuo ; Asakura, Tetsuo ; Takeda, Mitsuhiro ; Terauchi, Tsutomu ; Kainosho, Masatsune ; Ishii, Yoshitaka. / Nano-mole scale side-chain signal assignment by1H-detected protein solid-state NMR by ultra-fast magic-angle spinning and stereo-array isotope labeling. In: PLoS One. 2015 ; Vol. 10, No. 4.
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