Davis J H
Graduate Group in Biophysics, University of California, San Francisco 94143-0448, USA.
J Biomol NMR. 1995 Jun;5(4):433-7. doi: 10.1007/BF00182288.
2D 15N-1H correlation spectra are ideal for measuring backbone amide populations to determine amide exchange protection factors in studies of protein folding or other structural features. Most protein NMR spectroscopists use HSQC, which has been shown to be generally superior to HMQC in both resolution and sensitivity. The refocused HSQC experiment is intrinsically less sensitive than the regular HSQC, due to T2 relaxation during the refocusing delays. However, we show here that, when high 15N resolution is needed, an optimized refocused HSQC sequence that utilizes a semi-constant time evolution period and pulsed field gradients has better signal-to-noise ratio and resolution, and integrates more accurately, than a similar HSQC. The differences are demonstrated on a 20 kDa protein. The technique can also be applied to 3D NOESY experiments to eliminate strong NH2 geminal peaks and their truncation artefacts at a modest cost in sensitivity.
二维¹⁵N-¹H相关谱对于测量主链酰胺基团丰度以确定蛋白质折叠或其他结构特征研究中的酰胺交换保护因子而言是理想的。大多数蛋白质核磁共振光谱学家使用异核单量子相干谱（HSQC），在分辨率和灵敏度方面，它已被证明总体上优于异核多量子相干谱（HMQC）。由于在重聚焦延迟期间的横向弛豫，重聚焦HSQC实验本质上比常规HSQC灵敏度更低。然而，我们在此表明，当需要高¹⁵N分辨率时，与类似的HSQC相比，一种利用半恒定时间演化期和脉冲场梯度的优化重聚焦HSQC序列具有更好的信噪比和分辨率，并且积分更准确。在一个20 kDa的蛋白质上展示了这些差异。该技术还可应用于三维核欧沃豪斯效应谱（NOESY）实验，以消除强NH₂偕偶峰及其截断伪影，且灵敏度仅有适度损失。
