NMR Spectroscopy, Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands.
MOE Key Lab for Membrane-less Organelles & Cellular Dynamics, School of Life Sciences, University of Science and Technology of China, 96 Jinzhai Road, Hefei, 230026, Anhui, China.
Nat Commun. 2020 Jan 2;11(1):18. doi: 10.1038/s41467-019-13876-x.
Microtubules are important components of the eukaryotic cytoskeleton. Their structural organization is regulated by nucleotide binding and many microtubule-associated proteins (MAPs). While cryo-EM and X-ray crystallography have provided detailed views of interactions between MAPs with the microtubule lattice, little is known about how MAPs and their intrinsically disordered regions interact with the dynamic microtubule surface. NMR carries the potential to directly probe such interactions but so far has been precluded by the low tubulin yield. We present a protocol to produce [C, N]-labeled, functional microtubules (MTs) from human cells for solid-state NMR studies. This approach allowed us to demonstrate that MAPs can differently modulate the fast time-scale dynamics of C-terminal tubulin tails, suggesting distinct interaction modes. Our results pave the way for in-depth NMR studies of protein dynamics involved in MT assembly and their interactions with other cellular components.
微管是真核细胞骨架的重要组成部分。它们的结构组织受核苷酸结合和许多微管相关蛋白 (MAPs) 的调节。虽然冷冻电镜和 X 射线晶体学已经提供了 MAPs 与微管晶格之间相互作用的详细视图，但对于 MAPs 及其固有无序区域如何与动态微管表面相互作用知之甚少。NMR 有可能直接探测到这种相互作用，但到目前为止，由于微管蛋白产量低而受到限制。我们提出了一种从人类细胞中产生用于固态 NMR 研究的 [C, N]-标记功能微管 (MT) 的方案。这种方法使我们能够证明 MAPs 可以不同地调节 C 末端微管尾部的快速时间尺度动力学，表明存在不同的相互作用模式。我们的结果为深入研究 MT 组装过程中涉及的蛋白质动力学及其与其他细胞成分的相互作用铺平了道路。
