Optimum levels of exchangeable protons in perdeuterated proteins for proton detection in MAS solid-state NMR spectroscopy
Optimum levels of exchangeable protons in perdeuterated proteins for proton detection in MAS solid-state NMR spectroscopy
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DOI：
10.1007/s10858-009-9369-0
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发表时间：
2010-01-01
影响因子：
2.7
通讯作者：
Oschkinat, Hartmut
中科院分区：
文献类型：
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作者：
Akbey, Uemit;Lange, Sascha;Oschkinat, Hartmut
我们对可交换质子水平对在全氘代蛋白质中获得的酰胺质子线宽的影响进行了系统研究。将结晶缓冲液中使用的D₂O量从90%降低到0%时，我们观察到H - 1和N - 15共振的线宽都增加了四倍。同时，我们发现在基于偶极耦合的实验中，对于H₂O浓度高达40%的情况，H - 1 - N - 15相关性的信噪比（SNR）逐渐增加。超过40%时，观察到信噪比显著降低。基于标量耦合的H - 1 - N - 15相关性实验对于用≤30% H₂O制备的样品产生几乎恒定的信噪比。在结晶过程中使用更多H₂O的样品显示出核磁共振强度显著降低。通过考虑在含有更多质子的样品中H - 1 T₁的降低来计算信噪比（单位时间的信噪比），对于基于标量耦合和偶极耦合的实验，分别使用30%和40% H₂O结晶的样品获得最大信噪比。在基于交叉极化（CP）的实验中，将H₂O浓度从10%增加到40%可获得3.8的灵敏度增益，而线宽仅变宽1.5倍。一般来说，我们发现当可能的H - 1，H - 1相互作用数量增加时，与基于非灵敏核极化转移增强（INEPT）的转移相比，交叉极化（CP）更有利。在低氘代水平（结晶缓冲液中≥60% H₂O）时，刚性残基的共振变宽以至于无法检测。所有实验均在24 kHz的魔角旋转（MAS）频率下，使用鸡α - 血影蛋白SH3结构域的全氘代样品进行。
We present a systematic study of the effect of the level of exchangeable protons on the observed amide proton linewidth obtained in perdeuterated proteins. Decreasing the amount of D2O employed in the crystallization buffer from 90 to 0%, we observe a fourfold increase in linewidth for both H-1 and N-15 resonances. At the same time, we find a gradual increase in the signal-to-noise ratio (SNR) for H-1-N-15 correlations in dipolar coupling based experiments for H2O concentrations of up to 40%. Beyond 40%, a significant reduction in SNR is observed. Scalar-coupling based H-1-N-15 correlation experiments yield a nearly constant SNR for samples prepared with a parts per thousand currency sign30% H2O. Samples in which more H2O is employed for crystallization show a significantly reduced NMR intensity. Calculation of the SNR by taking into account the reduction in H-1 T (1) in samples containing more protons (SNR per unit time), yields a maximum SNR for samples crystallized using 30 and 40% H2O for scalar and dipolar coupling based experiments, respectively. A sensitivity gain of 3.8 is obtained by increasing the H2O concentration from 10 to 40% in the CP based experiment, whereas the linewidth only becomes 1.5 times broader. In general, we find that CP is more favorable compared to INEPT based transfer when the number of possible H-1,H-1 interactions increases. At low levels of deuteration (a parts per thousand yen60% H2O in the crystallization buffer), resonances from rigid residues are broadened beyond detection. All experiments are carried out at MAS frequency of 24 kHz employing perdeuterated samples of the chicken alpha-spectrin SH3 domain.