High-speed atomic force microscopy shows that annexin V stabilizes membranes on the second timescale
High-speed atomic force microscopy shows that annexin V stabilizes membranes on the second timescale
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DOI：
10.1038/nnano.2016.89
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发表时间：
2016-09-01
影响因子：
38.3
通讯作者：
Scheuring, Simon
中科院分区：
文献类型：
--
作者：
Miyagi, Atsushi;Chipot, Christophe;Scheuring, Simon
膜联蛋白是一种丰富的细胞质蛋白，可以以Ca2+依赖的方式与带负电荷的磷脂结合，并且已知在Ca2+的储存和膜愈合中发挥作用。然而，关于蛋白质- ca2 +膜组装和拆卸的动态过程知之甚少。在这里，我们展示了高速原子力显微镜（HS-AFM）可以用来反复诱导和破坏膜联蛋白组装，并研究它们的结构、动力学和相互作用。我们的HS-AFM装置通过集成用于缓冲交换的泵送系统和用于释放笼状化合物的脉冲激光系统，适用于此类生物应用。我们发现生物化学上相同的膜联蛋白（膜联蛋白V）根据组装位置显示不同的有效Ca2+和膜亲和力，在保持膜稳定性的同时提供广泛的Ca2+缓冲机制。我们还表明，膜联蛋白是膜募集的，并在5秒内形成稳定的超分子组装，其条件与细胞中的膜损伤相当。分子动力学模拟提供了Ca2+在膜联蛋白与膜表面的可逆结合中所起作用的原子细节。
Annexins are abundant cytoplasmic proteins that can bind to negatively charged phospholipids in a Ca2+-dependent manner, and are known to play a role in the storage of Ca2+ and membrane healing. Little is known, however, about the dynamic processes of protein-Ca2+-membrane assembly and disassembly. Here we show that high-speed atomic force microscopy (HS-AFM) can be used to repeatedly induce and disrupt annexin assemblies and study their structure, dynamics and interactions. Our HS-AFM set-up is adapted for such biological applications through the integration of a pumping system for buffer exchange and a pulsed laser system for uncaging caged compounds. We find that biochemically identical annexins (annexin V) display different effective Ca2+ and membrane affinities depending on the assembly location, providing a wide Ca2+ buffering regime while maintaining membrane stabilization. We also show that annexin is membrane-recruited and forms stable supramolecular assemblies within 5 s in conditions that are comparable to a membrane lesion in a cell. Molecular dynamics simulations provide atomic detail of the role played by Ca2+ in the reversible binding of annexin to the membrane surface.