Zhou Yuxuan, Zhang Beirong, Zhong Bowen, Sun Min, Zhang Xu, Zhang Yukui, Zhang Lihua, Zhao Qun, Gong Zhou, Liu Maili
State Key Laboratory of Magnetic Resonance Spectroscopy and Imaging, Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences, Wuhan, Hubei 430071, China.
University of Chinese Academy of Sciences, Beijing 100049, China.
JACS Au. 2025 Jul 9;5(7):3649-3659. doi: 10.1021/jacsau.5c00709. eCollection 2025 Jul 28.
Understanding protein structure and dynamics is crucial to elucidating their biological functions. Recent advancements in experimental techniques and AI-driven predictions, such as AlphaFold, have significantly enhanced our ability to determine protein structures. However, most methods are based on studies, which often fail to capture the complexities of protein behavior . This study focuses on chemical cross-linking mass spectrometry (XL-MS) as a promising approach to investigate protein structures within their native cellular environments. We systematically evaluate the performance of four commonly used cross-linkers, analyzing their structural characteristics, like length and dihedral angles, as well as the resulting thermodynamic and kinetic behaviors. By combining extensive XL-MS data with all-atom molecular dynamics simulations, we elucidate the relationship between cross-linker structure and their effectiveness in probing protein dynamics. The results reveal the relationship between the flexibility of cross-linkers and their structural characteristics and further elucidate that more rigid cross-linkers are able to capture more protein dynamic information, especially the dynamics of disordered regions. This study provides critical insights into cross-linker selection and design, offering a framework for future XL-MS applications aimed at advancing our understanding of protein dynamics and interactions in living cells.
了解蛋白质的结构和动力学对于阐明其生物学功能至关重要。实验技术和人工智能驱动的预测方法（如AlphaFold）的最新进展显著提高了我们确定蛋白质结构的能力。然而，大多数方法都基于研究，这些研究往往无法捕捉蛋白质行为的复杂性。本研究聚焦于化学交联质谱法（XL-MS），这是一种在天然细胞环境中研究蛋白质结构的有前景的方法。我们系统地评估了四种常用交联剂的性能，分析了它们的结构特征，如长度和二面角，以及由此产生的热力学和动力学行为。通过将大量的XL-MS数据与全原子分子动力学模拟相结合，我们阐明了交联剂结构与其探测蛋白质动力学有效性之间的关系。结果揭示了交联剂的灵活性与其结构特征之间的关系，并进一步阐明，更刚性的交联剂能够捕捉更多的蛋白质动态信息，尤其是无序区域的动态信息。本研究为交联剂的选择和设计提供了关键见解，为未来旨在推进我们对活细胞中蛋白质动力学和相互作用理解的XL-MS应用提供了一个框架。
