Le Nguyen Ngoc Lan, Pandey Ras B, Sompornpisut Pornthep
Center of Excellence in Computational Chemistry, Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand.
School of Mathematics and Natural Sciences, University of Southern Mississippi, Hattiesburg, Mississippi 39406, United States.
J Phys Chem B. 2021 Jan 28;125(3):748-756. doi: 10.1021/acs.jpcb.0c08993. Epub 2021 Jan 18.
The nitroxide spin label is the most widely used probe for electron paramagnetic resonance (EPR) spectroscopy studies of the structure and function of biomolecules. However, the role of surrounding environments in determining the dynamics of nitroxide spin labels in biological complex systems remains to be clarified. This study aims to characterize the dynamics and environmental structure of spin labels in the voltage-sensing domain (VSD) of a KvAP potassium channel by means of molecular dynamics (MD) studies. MD simulations for unlabeled and 132 spin-labeled KvAP-VSD models (spin labels introduced at positions 20-151) were carried out in a phospholipid bilayer to evaluate conformational dynamics of nitroxide spin-label side chains in the VSD. Structural flexibility, conformational freedom, and orientation of the spin-label side chains were investigated in relation to their dynamics in different microenvironments. The analysis of MD data showed that the attached spin-label probe did not severely perturb the protein dynamics. The conformational freedoms of the nitroxide side chain vary with the physical structure of the surrounding environments. The two terminal dihedral angles of the nitroxide side chain tend to cluster and adopt several preferred rotameric states. From the nearest-neighbor analysis, the spin label can be exposed to either a homogeneous or heterogeneous environment with various exposure scenarios. The dynamical movement of KvAP-VSD is high at a water-exposed site, moderate in the membrane, and low in the protein core. Understanding the structure and dynamics behaviors of spin labels helps to manage the experimental uncertainty and avoid misleading interpretation in relation to the protein structure.
氮氧自由基自旋标记是用于生物分子结构与功能的电子顺磁共振（EPR）光谱研究中使用最广泛的探针。然而，在生物复杂系统中，周围环境在决定氮氧自由基自旋标记动力学方面所起的作用仍有待阐明。本研究旨在通过分子动力学（MD）研究来表征KvAP钾通道电压感应域（VSD）中自旋标记的动力学和环境结构。在磷脂双层中对未标记的和132个自旋标记的KvAP-VSD模型（在第20至151位引入自旋标记）进行MD模拟，以评估VSD中氮氧自由基自旋标记侧链的构象动力学。研究了自旋标记侧链的结构灵活性、构象自由度及其在不同微环境中的动力学相关取向。MD数据分析表明，附着的自旋标记探针不会严重干扰蛋白质动力学。氮氧侧链的构象自由度随周围环境的物理结构而变化。氮氧侧链的两个末端二面角倾向于聚集并采用几种优选的旋转异构体状态。通过最近邻分析，自旋标记可能会暴露于具有各种暴露情况的均匀或异质环境中。KvAP-VSD在水暴露位点的动态运动较高，在膜中适中，在蛋白质核心中较低。了解自旋标记的结构和动力学行为有助于控制实验不确定性，并避免在蛋白质结构方面产生误导性解释。
