Center for Biophysics and Quantitative Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA.
Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL, USA.
Phys Chem Chem Phys. 2023 Feb 22;25(8):6253-6262. doi: 10.1039/d2cp02890e.
Spectroscopy experiments are crucial to study membrane proteins for which traditional structure determination methods still prove challenging. Double electron-electron resonance (DEER) spectroscopy experiments provide protein residue-pair distance distributions that are indicative of their conformational heterogeneity. Atomistic molecular dynamics (MD) simulations are another tool that have been proven to be vital to study the structural dynamics of membrane proteins such as to identify inward-open, occluded, and outward-open conformations of transporter membrane proteins, among other partially open or closed states of the protein. Yet, studies have reported that there is no direct consensus between the distributional data from DEER experiments and MD simulations, which has challenged validation of structures obtained from long-timescale simulations and using simulations to design experiments. Current coping strategies for comparisons rely on heuristics, such as mapping the nearest matching peaks between two ensembles or biased simulations. Here we examine the differences in residue-pair distance distributions arising due to the choice of membranes around the protein and covalent modification of a pair of residues to nitroxide spin labels in DEER experiments. Through comparing MD simulations of two proteins, PepT and LeuT-both of which have been characterized using DEER experiments previously-we show that the proteins' dynamics are similar despite the choice of the detergent micelle as a membrane mimetic in DEER experiments. On the other hand, covalently modified residues show slight local differences in their dynamics and a huge divergence when the oxygen atom pair distances between spin labeled residues are measured rather than protein backbone distances. Given the computational expense associated with pairwise MTSSL labeled MD simulations, we examine the use of biased simulations to explore the conformational dynamics of the spin labels only to reveal that such simulations alter the underlying protein dynamics. Our study identifies the main cause for the mismatch between DEER experiments and MD simulations and will accelerate the development of potential mitigation strategies to improve the match.
光谱实验对于研究传统结构测定方法仍然具有挑战性的膜蛋白至关重要。双电子电子共振（DEER）光谱实验提供了蛋白质残基对距离分布，这表明其构象异质性。原子分子动力学（MD）模拟是另一种已被证明对研究膜蛋白结构动力学至关重要的工具，例如确定转运蛋白膜蛋白的内向开放、闭塞和外向开放构象，以及蛋白质的其他部分开放或关闭状态。然而，研究报告称，DEER 实验和 MD 模拟的分布数据之间没有直接共识，这挑战了从长时间尺度模拟获得的结构的验证，并使用模拟来设计实验。目前，比较的应对策略依赖于启发式方法，例如在两个集合之间映射最近匹配的峰或有偏差的模拟。在这里，我们研究了由于蛋白质周围膜的选择以及 DEER 实验中一对残基的共价修饰导致的残基对距离分布的差异。通过比较两种蛋白质（PepT 和 LeuT）的 MD 模拟，这两种蛋白质以前都使用 DEER 实验进行了表征，我们表明尽管在 DEER 实验中选择去污剂胶束作为膜模拟物，但蛋白质的动力学是相似的。另一方面，共价修饰的残基在其动力学上表现出轻微的局部差异，并且当测量自旋标记残基之间的氧原子对距离而不是蛋白质骨架距离时，它们的动力学存在巨大差异。鉴于与成对 MTSSL 标记 MD 模拟相关的计算费用，我们研究了使用有偏差的模拟来探索自旋标记的构象动力学，以揭示这种模拟会改变潜在的蛋白质动力学。我们的研究确定了 DEER 实验和 MD 模拟之间不匹配的主要原因，并将加速开发潜在的缓解策略以提高匹配度。
