Site-specific analysis of heteronuclear Overhauser effects in microcrystalline proteins
Site-specific analysis of heteronuclear Overhauser effects in microcrystalline proteins
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DOI：
10.1007/s10858-014-9843-1
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发表时间：
2014-07
影响因子：
2.7
通讯作者：
J. L. Amo;Vipin Agarwal;R. Sarkar;J. Porter;S. Asami;M. Rübbelke;U. Fink;Yi Xue;Oliver F. Lange;B. Reif
中科院分区：
文献类型：
--
作者：
J. L. Amo;Vipin Agarwal;R. Sarkar;J. Porter;S. Asami;M. Rübbelke;U. Fink;Yi Xue;Oliver F. Lange;B. Reif
诸如纵向弛豫的弛豫参数易受诸如自旋扩散的伪影的影响，并且可能受顺磁性杂质（例如氧）的影响，这使得定量解释变得困难。我们在这里提出的[1H] 13 C和[1H] 15 N在固定化蛋白质中的杂合率的位点特异性测量。对于甲基，由于甲基的三重旋转，预期会产生强烈的影响。[1H] 13 C杂合NOE结合13 C-R1的定量可以得到侧链运动参数的更准确的分析。某些主链酰胺以及特定天冬酰胺/谷氨酰胺侧链酰胺的显著[1H] 15 N杂环NOE的观察结果与MD模拟一致。通过使用高度氘代的微晶蛋白质样品来实现位点特异性杂原子NOE的测量，其中与质子化样品相比，自旋扩散减少。
Relaxation parameters such as longitudinal relaxation are susceptible to artifacts such as spin diffusion, and can be affected by paramagnetic impurities as e.g. oxygen, which make a quantitative interpretation difficult. We present here the site-specific measurement of [1H]13C and [1H]15N heteronuclear rates in an immobilized protein. For methyls, a strong effect is expected due to the three-fold rotation of the methyl group. Quantification of the [1H]13C heteronuclear NOE in combination with13C-R1can yield a more accurate analysis of side chain motional parameters. The observation of significant [1H]15N heteronuclear NOEs for certain backbone amides, as well as for specific asparagine/glutamine sidechain amides is consistent with MD simulations. The measurement of site-specific heteronuclear NOEs is enabled by the use of highly deuterated microcrystalline protein samples in which spin diffusion is reduced in comparison to protonated samples.