Transporting individual molecules across cell membranes is a fundamental process in cellular metabolism. Although the crystal diffraction technique has greatly contributed to our understanding of the structures of the involved transporters, a description of the dynamic transport mechanism at the single-molecule level has been extremely elusive. In this study, we applied atomic force microscopy (AFM)-based force tracing to directly monitor the transport of a single molecule, D-glucose, across living cell membranes. Our results show that the force to transport a single molecule of D-glucose across cell membranes is 37 ± 9 pN, and the corresponding transport interval is approximately 20 ms, while the average speed is approximately 0.3 μm s⁻¹. Furthermore, our calculated force profile from molecular dynamics simulations showed quantitatively good agreement with the force tracing observation and revealed detailed information regarding the glucose transport path, indicating that two salt bridges, K38/E299 and K300/E426, play critical roles during glucose transport across glucose transporter 1 (GLUT1). This role was further verified using biological experiments that disrupted these two bridges and measured the uptake of glucose into the cells. Our approaches led to the first unambiguous description of the glucose transport process across cell membranes at the single-molecule level and demonstrated the biological importance of the two salt bridges for transporting glucose across GLUT1.

中文翻译：

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力追踪和分子动力学模拟揭示了单个葡萄糖分子的运输过程†

跨细胞膜转运单个分子是细胞代谢的基本过程。尽管晶体衍射技术极大地有助于我们理解所涉及的转运蛋白的结构，但是在单分子水平上对动态转运机理的描述却极为复杂。在这项研究中，我们应用了基于原子力显微镜（AFM）的力追踪技术来直接监控单个分子D-葡萄糖在活细胞膜上的转运。我们的结果表明，跨细胞膜转运单分子D-葡萄糖的力为37±9 pN，相应的转运间隔约为20 ms，而平均速度约为0.3μms ^-1。此外，我们从分子动力学模拟计算出的力分布图与力追踪观察结果在定量上吻合良好，并揭示了有关葡萄糖转运路径的详细信息，这表明两个盐桥K38 / E299和K300 / E426在葡萄糖转运过程中起着至关重要的作用。葡萄糖转运蛋白1（GLUT1）。使用破坏了这两个桥并测量葡萄糖向细胞中摄取的生物学实验，进一步证实了这一作用。我们的方法首次明确描述了单分子水平跨细胞膜的葡萄糖转运过程，并证明了两个盐桥对于跨GLUT1转运葡萄糖的生物学重要性。