Optimal degree of protonation for 1H detection of aliphatic sites in randomly deuterated proteins as a function of the MAS frequency
Optimal degree of protonation for 1H detection of aliphatic sites in randomly deuterated proteins as a function of the MAS frequency
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DOI：
10.1007/s10858-012-9659-9
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发表时间：
2012-10-01
影响因子：
2.7
通讯作者：
Reif, Bernd
中科院分区：
文献类型：
--
作者：
Asami, Sam;Szekely, Kathrin;Reif, Bernd
H-1偶极网络，这是在固态中应用质子检测的主要障碍，可以通过氘化来减少，采用RAP（减少的邻接质子化）标记方案，其在不可交换位点产生随机质子化。我们在这里提出了一个系统的研究RAP样品中的随机侧链质子化的最佳程度作为MAS（魔角自旋）频率的函数。特别是，我们比较了H-1的敏感性和线宽的微晶蛋白，鸡α-血影蛋白的SH 3结构域，样品，制备5- 25%的水在E。coli生长培养基中，在20-60 kHz的MAS频率范围内。在19.96 T（850 MHz）的外场下，我们发现在M9介质中使用15%至25%的质子浓度在灵敏度和分辨率方面产生最佳折衷，可实现的平均H-1线宽为40-50 Hz。比较在MAS频率为60与20 kHz时的灵敏度，在基于INEPT的H-1检测到的1D H-1，C-13相关实验中观察到灵敏度增加了4-4.5倍。总的来说，我们发现在60 kHz下用1.3 mm转子记录的光谱与在20 kHz下用完全填充的3.2 mm转子记录的光谱具有几乎相同的灵敏度，即使使用的材料少20倍。灵敏度的提高归因于快速MAS导致的H-1线变窄以及1.3 mm线圈的效率提高。
The H-1 dipolar network, which is the major obstacle for applying proton detection in the solid-state, can be reduced by deuteration, employing the RAP (Reduced Adjoining Protonation) labeling scheme, which yields random protonation at non-exchangeable sites. We present here a systematic study on the optimal degree of random sidechain protonation in RAP samples as a function of the MAS (magic angle spinning) frequency. In particular, we compare H-1 sensitivity and linewidth of a microcrystalline protein, the SH3 domain of chicken alpha-spectrin, for samples, prepared with 5-25 % H2O in the E. coli growth medium, in the MAS frequency range of 20-60 kHz. At an external field of 19.96 T (850 MHz), we find that using a proton concentration between 15 and 25 % in the M9 medium yields the best compromise in terms of sensitivity and resolution, with an achievable average H-1 linewidth on the order of 40-50 Hz. Comparing sensitivities at a MAS frequency of 60 versus 20 kHz, a gain in sensitivity by a factor of 4-4.5 is observed in INEPT-based H-1 detected 1D H-1,C-13 correlation experiments. In total, we find that spectra recorded with a 1.3 mm rotor at 60 kHz have almost the same sensitivity as spectra recorded with a fully packed 3.2 mm rotor at 20 kHz, even though similar to 20x less material is employed. The improved sensitivity is attributed to H-1 line narrowing due to fast MAS and to the increased efficiency of the 1.3 mm coil.