Structure of fully protonated proteins by proton-detected magic-angle spinning NMR
Structure of fully protonated proteins by proton-detected magic-angle spinning NMR
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DOI：
10.1073/pnas.1602248113
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发表时间：
2016-08-16
影响因子：
11.1
通讯作者：
Pintacuda, Guido
中科院分区：
文献类型：
--
作者：
Andreas, Loren B.;Jaudzems, Kristaps;Pintacuda, Guido
通过质子检测魔角旋转（MAS）NMR的蛋白质结构测定集中在高度氘代的样品上，其中仅引入少量质子，并且从侧链观察到的信号极其有限。在这里，我们表明，在两个完全质子化的蛋白质，在100 kHz MAS及以上，光谱分辨率是足够高，以检测解决所有残基类型的酰胺和侧链质子的相关性，并可靠地测量一个密集的网络H-1-H-1的邻近定义的蛋白质结构。高数据质量允许通过自动数据分析正确识别编码在3D光谱中的核间距离约束，从而实现准确、无偏和快速的结构测定。此外，我们发现，较窄的质子共振线，较长的相干寿命，和改善的磁化转移抵消在100 kHz以上的自旋减少的样品尺寸。少于2周的实验时间和一个单一的0.5毫克的样品是足够的采集所有必要的数据的骨干和侧链共振分配和无监督的结构测定。我们期望该技术为适用于广泛蛋白质的原子分辨率结构分析铺平道路。
Protein structure determination by proton-detected magic-angle spinning (MAS) NMR has focused on highly deuterated samples, in which only a small number of protons are introduced and observation of signals from side chains is extremely limited. Here, we show in two fully protonated proteins that, at 100-kHz MAS and above, spectral resolution is high enough to detect resolved correlations from amide and side-chain protons of all residue types, and to reliably measure a dense network of H-1-H-1 proximities that define a protein structure. The high data quality allowed the correct identification of internuclear distance restraints encoded in 3D spectra with automated data analysis, resulting in accurate, unbiased, and fast structure determination. Additionally, we find that narrower proton resonance lines, longer coherence lifetimes, and improved magnetization transfer offset the reduced sample size at 100-kHz spinning and above. Less than 2 weeks of experiment time and a single 0.5-mg sample was sufficient for the acquisition of all data necessary for backbone and side-chain resonance assignment and unsupervised structure determination. We expect the technique to pave the way for atomic-resolution structure analysis applicable to a wide range of proteins.