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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

机译:通过超快幻角旋转和立体阵列同位素标记的1H检测到的蛋白质固态NMR进行纳米级旁链信号分配

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摘要

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.
机译:我们提出了一种结合1H检测的13C固态NMR(SSNMR)的通用方法,以结合使用高磁场,超快速魔角旋转(MAS)的10-50 nmol量的蛋白质的侧链信号分配频率约为80 kHz,并带有立体阵列同位素标记(SAIL)蛋白[Kainosho M.等人,Nature 440,52-57,2006]。首先,我们证明1H间接检测可提高SAIL蛋白在超快MAS条件下侧链分配的13C SSNMR灵敏度和分辨率。对在7个异亮氨酸(Ile)残基处进行SAIL标记的微晶遍在蛋白(〜55 nmol或〜0.5mg)进行1H检测的SSNMR。 1H检测的2D 1H / 13C SSNMR的灵敏度分别比13C检测的2D 1H / 13C SSNMR和1D 13C CPMAS分别提高了5.4-9.7和2.1-5.0,证明了2D 1H检测的SSNMR不仅提供了首次获得了比1D 13C检测更高的分辨率和灵敏度优势。 SAIL标记的侧链残基的高1H分辨率即使在2D数据中也提供了合理的分辨率。在SAIL泛素上进行的1H检测的3D 13C / 13C / 1H实验仅在约2.5小时内就为七个Ile残基提供了几乎完整的1H和13C分配。结果表明,在约3天内,仅用10 nmol的蛋白质样品,采用这种方法进行侧链信号分配的可行性。该方法可能适用于多种生物学感兴趣的蛋白质,而无需高效蛋白质表达系统的任何要求。

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