Buffer effects on site directed Cu2+-labeling using the double histidine motif
Buffer effects on site directed Cu2+-labeling using the double histidine motif
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DOI：
10.1016/j.jmr.2020.106848
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发表时间：
2020-11-01
影响因子：
2.2
通讯作者：
Saxena, Sunil
中科院分区：
文献类型：
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作者：
Jarvi, Austin Gamble;Casto, Joshua;Saxena, Sunil
双组氨酸基序（dHis）已经成为一种强大的自旋标记工具，可以通过基于Cu2+的电子顺磁共振来确定蛋白质的构象和动力学、亚基取向、天然金属结合位点位置和其他物理特性。在这里，我们研究了该技术在五种常见缓冲系统中的有效性，并表明缓冲系统的选择会影响Cu2+-NTA在dHis位点上的负载，更一般地说，会影响整个技术的灵敏度。我们还提出了一种标准化和优化的方法，可以用Cu2+-NTA标记dHis基序，用于基于EPR的距离测量。我们提供最佳的装载程序，使用代表性的EPR和UV/Vis数据为过程中的每个步骤。从这些数据中，我们发现低温样品孵育可以在30分钟内达到最大dHis负载。仅使用这些最佳程序，我们看到与先前发表的n -乙基morpholine结果相比，完全标记的蛋白质增加了28%。使用这两种优化的程序以及更优化的缓冲液，我们可以获得高达80%的满载蛋白，与之前的数据相比，这相当于增加了64%。这些结果提供了对dHis Cu2+-NTA系统，影响其功效的变量的深入了解，并提出了一种方法，通过该方法可以减轻这些问题，以获得最有效的标记。(C) 2020爱思唯尔公司版权所有。
The double histidine, or dHis, motif has emerged as a powerful spin labeling tool to determine the conformations and dynamics, subunit orientation, native metal binding site location, and other physical characteristics of proteins by Cu2+-based electron paramagnetic resonance. Here, we investigate the efficacy of this technique in five common buffer systems, and show that buffer choice can impact the loading of Cu2+-NTA into dHis sites, and more generally, the sensitivity of the overall technique. We also present a standardized and optimized examination of labeling of the dHis motif with Cu2+-NTA for EPR based distance measurements. We provide optimal loading procedures, using representative EPR and UV/Vis data for each step in the process. From this data, we find that maximal dHis loading can be achieved in under 30 min with low temperature sample incubation. Using only these optimal procedures, we see up to a 28% increase in fully labeled proteins compared to previously published results in N-ethylmorpholine. Using both this optimized procedure as well as a more optimal buffer, we can achieve up to 80% fully loaded proteins, which corresponds to a 64% increase compared to the prior data. These results provide insight and deeper understanding of the dHis Cu2+-NTA system, the variables that impact its efficacy, and present a method by which these issues may be mitigated for the most efficient labeling. (C) 2020 Elsevier Inc. All rights reserved.