Beyond structure: Deciphering site‐specific dynamics in proteins from double histidine‐based EPR measurements
Beyond structure: Deciphering site‐specific dynamics in proteins from double histidine‐based EPR measurements
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DOI：
10.1002/pro.4359
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发表时间：
2022-06
期刊：
影响因子：
8
通讯作者：
Kevin Singewald;James A. Wilkinson;Zikri Hasanbasri;S. Saxena
中科院分区：
文献类型：
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作者：
Kevin Singewald;James A. Wilkinson;Zikri Hasanbasri;S. Saxena
蛋白质的位点特异性动力学是蛋白质功能的核心。虽然电子顺磁共振 (EPR) 具有测量大型蛋白质复合物动力学的潜力，但对柔性硝基氧标记的依赖受到限制，尤其是对于位点特异性 β-折叠动力学的精确测量。在这里，我们使用 EPR 光谱来测量蛋白质 GB1 表面的位点特异性动态。通过使用双组氨酸 (dHis) 基序，可以使用 Cu(II) - 次氮基三乙酸 (NTA) 复合物进行标记，获得了 α 螺旋和 β 折叠位点的动态信息。由此产生的 CW-EPR 光谱模拟报告了 GB1 表面独特的特定位点波动。此外，我们还进行了分子动力学 (MD) 模拟来补充 EPR 数据。从 MD 观察到的动态与 EPR 结果一致。此外，我们观察到不同站点的 gǁ 值存在微小变化，这可能是由于站点的配位几何和/或局部静电的微小差异所致。总而言之，这项工作扩展了基于 Cu(II)NTA 的 EPR 测量的实用性，以探测超出距离限制的信息。
Site‐specific dynamics in proteins are at the heart of protein function. While electron paramagnetic resonance (EPR) has potential to measure dynamics in large protein complexes, the reliance on flexible nitroxide labels is limitating especially for the accurate measurement of site‐specific β‐sheet dynamics. Here, we employed EPR spectroscopy to measure site‐specific dynamics across the surface of a protein, GB1. Through the use of the double Histidine (dHis) motif, which enables labeling with a Cu(II) – nitrilotriacetic acid (NTA) complex, dynamics information was obtained for both α‐helical and β‐sheet sites. Spectral simulations of the resulting CW‐EPR report unique site‐specific fluctuations across the surface of GB1. Additionally, we performed molecular dynamics (MD) simulations to complement the EPR data. The dynamics observed from MD agree with the EPR results. Furthermore, we observe small changes in gǁ values for different sites, which may be due to small differences in coordination geometry and/or local electrostatics of the site. Taken together, this work expands the utility of Cu(II)NTA‐based EPR measurements to probe information beyond distance constraints.