Quantifying Microsecond Exchange in Large Protein Complexes with Accelerated Relaxation Dispersion Experiments in the Solid State
Quantifying Microsecond Exchange in Large Protein Complexes with Accelerated Relaxation Dispersion Experiments in the Solid State
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DOI：
10.1038/s41598-019-47507-8
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发表时间：
2019-07-31
影响因子：
4.6
通讯作者：
Lewandowski, Jozef R.
中科院分区：
文献类型：
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作者：
Oster, Carl;Kosol, Simone;Lewandowski, Jozef R.
固体核磁共振是获得蛋白质复合物结构和动力学信息的有力方法，由于溶解度和大小的限制，这些信息是其他方法无法获得的。在这里，我们提出了一种方法，可以通过使用顺磁剂来加速N-15 R-1p弛豫色散测量并克服灵敏度限制，从而量化大型蛋白质复合物中的微秒构象交换。该方法在结晶GB1上验证，然后应用于GB1与全长人免疫球蛋白G （IgG）沉淀的> 300 kDa复合物。顺磁剂的加入使每单位时间内的信噪比增加了5倍，这使得850和700 MHz光谱仪在5天和10天内分别在含有少于50 μ g标记材料的样品上记录了完整的弛豫色散曲线。我们发现在GB1 in晶体和IgG in复合物的β -片上也有类似的交换过程。然而，在晶体GB1的α -地狱层中观察到的一些残基的缓慢运动在配合物中没有检测到。
Solid state NMR is a powerful method to obtain information on the structure and dynamics of protein complexes that, due to solubility and size limitations, cannot be achieved by other methods. Here, we present an approach that allows the quantification of microsecond conformational exchange in large protein complexes by using a paramagnetic agent to accelerate N-15 R-1p relaxation dispersion measurements and overcome sensitivity limitations. The method is validated on crystalline GB1 and then applied to a > 300 kDa precipitated complex of GB1 with full length human immunoglobulin G (IgG).The addition of a paramagnetic agent increased the signal to noise ratio per time unit by a factor of 5, which allowed full relaxation dispersion curves to be recorded on a sample containing less than 50 mu g of labelled material In 5 and 10 days on 850 and 700 MHz spectrometers, respectively. We discover a similar exchange process across the beta-sheet in GB1 In crystals and In complex with IgG. However, the slow motion observed fora number of residues in the alpha-hellx of crystalline GB1 Is not detected In the complex.