Intrinsically disordered proteins (IDPs) often contain proline residues that undergo cis/trans isomerization. While molecular dynamics (MD) simulations have the potential to fully characterize the proline cis and trans subensembles, they are limited by the slow timescales of isomerization and force field inaccuracies. NMR spectroscopy can report on ensemble-averaged observables for both the cis-proline and trans-proline states, but a full atomistic characterization of these conformers is challenging. Given the importance of proline cis/trans isomerization for influencing the conformational sampling of disordered proteins, we employed a combination of all-atom MD simulations with enhanced sampling (metadynamics), NMR, and small-angle x-ray scattering (SAXS) to characterize the two subensembles of the ORF6 C-terminal region (ORF6CTR) from SARS-CoV-2 corresponding to the proline-57 (P57) cis and trans states. We performed MD simulations in three distinct force fields: AMBER03ws, AMBER99SB-disp, and CHARMM36m, which are all optimized for disordered proteins. Each simulation was run for an accumulated time of 180–220 μs until convergence was reached, as assessed by blocking analysis. A good agreement between the cis-P57 populations predicted from metadynamic simulations in AMBER03ws was observed with populations obtained from experimental NMR data. Moreover, we observed good agreement between the radius of gyration predicted from the metadynamic simulations in AMBER03ws and that measured using SAXS. Our findings suggest that both the cis-P57 and trans-P57 conformations of ORF6CTR are extremely dynamic and that interdisciplinary approaches combining both multiscale computations and experiments offer avenues to explore highly dynamic states that cannot be reliably characterized by either approach in isolation.

中文翻译：

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无序肽中 proline cis 和 trans 构象异构体的整合表征


固有无序蛋白 （IDP） 通常包含经历顺式/反式异构化的脯氨酸残基。虽然分子动力学 （MD） 模拟有可能完全表征脯氨酸、顺式和反子系，但它们受到异构化和力场不准确的缓慢时间尺度的限制。NMR 波谱可以报告顺式脯氨酸和反脯氨酸态的集合平均可观测物，但对这些构象异构体进行完整的原子表征是具有挑战性的。鉴于脯氨酸顺式/反式异构化对影响无序蛋白质构象采样的重要性，我们采用了全原子 MD 模拟与增强采样（宏动力学）、NMR 和小角 X 射线散射 （SAXS） 的组合来表征 SARS-CoV-2 的 ORF6 C 末端区域 （ORF6CTR） 的两个子系，对应于脯氨酸 57 （P57） 顺式和反式状态。我们在三个不同的力场中进行了 MD 模拟： AMBER03ws、 AMBER99SB-disp 和 CHARMM36m，它们都针对无序蛋白质进行了优化。每个模拟运行了 180-220 μs 的累积时间，直到达到收敛，如分块分析所评估的那样。观察到从 AMBER03ws 的元动力学模拟预测的 cis-P57 种群与从实验 NMR 数据中获得的种群之间具有良好的一致性。此外，我们观察到从 AMBER03ws 中的元动力学模拟预测的回转半径与使用 SAXS 测量的回转半径之间具有良好的一致性。 我们的研究结果表明，ORF6CTR 的 cis-P57 和 trans-P57 构象都是非常动态的，并且结合多尺度计算和实验的跨学科方法为探索高度动态状态提供了途径，这些状态无法单独通过任何一种方法进行可靠表征。