Bibliographic Details
| 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. |
| Authors: |
Songlin Wang, Sudhakar Parthasarathy, Yusuke Nishiyama, Yuki Endo, Takahiro Nemoto, Kazuo Yamauchi, Tetsuo Asakura, Mitsuhiro Takeda, Tsutomu Terauchi, Masatsune Kainosho, Yoshitaka Ishii |
| Source: |
PLoS ONE, Vol 10, Iss 4, p e0122714 (2015) |
| Publisher Information: |
Public Library of Science (PLoS), 2015. |
| Publication Year: |
2015 |
| Collection: |
LCC:Medicine
LCC:Science |
| Subject Terms: |
Medicine, Science |
| More Details: |
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. |
| Document Type: |
article |
| File Description: |
electronic resource |
| Language: |
English |
| ISSN: |
1932-6203 |
| Relation: |
http://europepmc.org/articles/PMC4391754?pdf=render; https://doaj.org/toc/1932-6203 |
| DOI: |
10.1371/journal.pone.0122714 |
| Access URL: |
https://doaj.org/article/7adf7229f38e4ad1aa73d1733a841450 |
| Accession Number: |
edsdoj.7adf7229f38e4ad1aa73d1733a841450 |
| Database: |
Directory of Open Access Journals |
