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.
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...
| Main Authors: | , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2015-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC4391754?pdf=render |
| _version_ | 1818298033543053312 |
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| author | Songlin Wang Sudhakar Parthasarathy Yusuke Nishiyama Yuki Endo Takahiro Nemoto Kazuo Yamauchi Tetsuo Asakura Mitsuhiro Takeda Tsutomu Terauchi Masatsune Kainosho Yoshitaka Ishii |
| author_facet | Songlin Wang Sudhakar Parthasarathy Yusuke Nishiyama Yuki Endo Takahiro Nemoto Kazuo Yamauchi Tetsuo Asakura Mitsuhiro Takeda Tsutomu Terauchi Masatsune Kainosho Yoshitaka Ishii |
| author_sort | Songlin Wang |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-13T04:28:54Z |
| format | Article |
| id | doaj.art-7adf7229f38e4ad1aa73d1733a841450 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-13T04:28:54Z |
| publishDate | 2015-01-01 |
| publisher | Public Library of Science (PLoS) |
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| series | PLoS ONE |
| spelling | doaj.art-7adf7229f38e4ad1aa73d1733a8414502022-12-21T23:59:38ZengPublic Library of Science (PLoS)PLoS ONE1932-62032015-01-01104e012271410.1371/journal.pone.0122714Nano-mole scale side-chain signal assignment by 1H-detected protein solid-state NMR by ultra-fast magic-angle spinning and stereo-array isotope labeling.Songlin WangSudhakar ParthasarathyYusuke NishiyamaYuki EndoTakahiro NemotoKazuo YamauchiTetsuo AsakuraMitsuhiro TakedaTsutomu TerauchiMasatsune KainoshoYoshitaka IshiiWe 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.http://europepmc.org/articles/PMC4391754?pdf=render |
| spellingShingle | Songlin Wang Sudhakar Parthasarathy Yusuke Nishiyama Yuki Endo Takahiro Nemoto Kazuo Yamauchi Tetsuo Asakura Mitsuhiro Takeda Tsutomu Terauchi Masatsune Kainosho Yoshitaka Ishii 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. PLoS ONE |
| title | 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. |
| title_full | 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. |
| title_fullStr | 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. |
| title_full_unstemmed | 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. |
| title_short | 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. |
| title_sort | 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 |
| url | http://europepmc.org/articles/PMC4391754?pdf=render |
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