Probing Surface Accessibility of Proteins Using Paramagnetic Relaxation in Solid-State NMR Spectroscopy
Probing Surface Accessibility of Proteins Using Paramagnetic Relaxation in Solid-State NMR Spectroscopy
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DOI：
10.1021/ja903892j
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发表时间：
2009-09-30
影响因子：
15
通讯作者：
Reif, Bernd
中科院分区：
文献类型：
--
作者：
Linser, Rasmus;Fink, Uwe;Reif, Bernd
顺磁弛豫增强（PRE）可以通过减少质子在固体状态下的纵向弛豫时间T来加速核磁共振数据的采集。我们发现，除了局部动力学外，散装溶剂中顺磁性化合物的存在还诱导了位点特异性弛豫，这取决于蛋白质中各自酰胺质子的表面可及性。通过比较不同浓度Cu-II（edta）螯合物制备的微晶蛋白样品的H-1 T-1倍，可以区分顺磁弛豫和动力学。我们发现羟基还可以介导弛豫，羟基通过与快速弛豫的散装溶剂交换的能力传递弛豫。此外，弛豫似乎是通过水分子转移的，水分子扩散到蛋白质结构中，并在蛋白质的柔性区域产生有效的差异PRE。实验是用含有90% D2O的缓冲液微晶化的α -光谱SH3结构域的氘化样品来证明的。氘化是避免自旋扩散的先决条件，否则会损害特定位点的分辨率。
Paramagnetic Relaxation Enhancement (PRE) can be used to accelerate NMR data acquisition by reducing the longitudinal proton relaxation time T, in the solid state. We show that the presence of paramagnetic compounds in the bulk solvent induces a site-specific relaxation in addition to local dynamics, which is dependent on the surface accessibility of the respective amide proton in the protein. Differentiation between paramagnetic relaxation and dynamics was achieved by a comparison of H-1 T-1 times obtained from microcrystalline protein samples prepared with different concentrations of the Cu-II(edta) chelate. We find that relaxation can in addition be mediated by hydroxyl groups, which transfer relaxation by their ability to exchange with the quickly relaxing bulk solvent. Furthermore, relaxation seems to be transferred by water molecules which diffuse into the protein structure and yield an efficient difference PRE in flexible regions of the protein. The experiments are demonstrated using a perdeuterated sample of the alpha-spectrin SH3 domain, which was microcrystallized from a buffer containing 90% D2O. Deuteration is a prerequisite to avoid spin diffusion which would otherwise compromise site specific resolution.