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.
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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DOI：
10.1371/journal.pone.0122714
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Ishii Y
中科院分区：
文献类型：
--
作者：
Wang S;Parthasarathy S;Nishiyama Y;Endo Y;Nemoto T;Yamauchi K;Asakura T;Takeda M;Terauchi T;Kainosho M;Ishii Y
我们提出了一种在1H检测的13 C固态NMR（SSNMR）中使用高磁场、~80 kHz的超快魔角旋转（MAS）和立体阵列同位素标记（SAIL）蛋白质的组合进行10-50 nmol量的蛋白质的侧链信号分配的一般方法[Kainosho M.例如，Nature 440，52-57，2006]。首先，我们证明，1H间接检测提高了灵敏度和分辨率的13 C SSNMR的SAIL蛋白的侧链分配在超快MAS条件。对在7个异亮氨酸（Ile）残基处SAIL标记的微晶泛素（约55 nmol或约0.5 mg）进行1H检测SSNMR。1H检测的2D 1H/13 C SSNMR的灵敏度比13 C检测的2D 1H/13 C SSNMR和1D 13 C CPMAS分别提高了5.4-9.7和2.1-5.0倍，表明2D 1H检测的SSNMR不仅提供了额外的分辨率，而且首次提供了比1D 13 C检测更高的灵敏度优势。即使在2D数据中，SAIL标记的侧链残基的高1H分辨率也提供了合理的分辨率。SAIL-泛素的1H-检测3D 13 C/13 C/1H实验仅在约2.5 h内提供了7个Ile残基的几乎完整的1H和13 C归属。结果表明，在这种方法中的侧链信号分配的可行性低至10 nmol的蛋白质样品在~3天内。该方法可能适用于多种生物学目的蛋白质，而不需要高效的蛋白质表达系统。
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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影响因子：
3.3
作者：
Huber, Matthias;Boeckmann, Anja;Meier, Beat H.
通讯作者：
Meier, Beat H.
影响因子：
16.6
作者：
Kobayashi, Takeshi;Mao, Kanmi;Pruski, Marek
通讯作者：
Pruski, Marek
影响因子：
4.4
作者：
Ishii, Y
通讯作者：
Ishii, Y
影响因子：
2.2
作者：
Ishii, Y;Tycko, R
通讯作者：
Tycko, R
影响因子：
2.7
作者：
DELAGLIO, F;GRZESIEK, S;BAX, A
通讯作者：
BAX, A