Direct observation of tropomyosin binding to actin filaments.
Direct observation of tropomyosin binding to actin filaments.
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直接观察原肌球蛋白与肌动蛋白丝的结合。
DOI：
10.1002/cm.21225
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发表时间：
2015
期刊：
影响因子：
--
通讯作者：
Moore,JeffreyR
中科院分区：
文献类型：
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作者：
Schmidt,WilliamM;Lehman,William;Moore,JeffreyR
原肌球蛋白是一种细长的α -螺旋盘绕线圈，沿着肌动蛋白丝的长螺距螺旋与七个连续的肌动蛋白亚基结合。一旦结合，原肌凝蛋白端对端聚合，既稳定肌动蛋白，又调节各种肌动蛋白结合蛋白（包括肌凝蛋白）在肌肉和非肌肉细胞中的通路。单个原肌凝蛋白分子与F - actin的结合较弱，具有milliolarkd，而端对端连接的原肌凝蛋白与F - actin的结合具有约1000倍的亲和力。尽管经过多年的研究，原肌凝蛋白在肌动蛋白丝上的组装机制仍不清楚。在这项研究中，我们使用全内反射荧光显微镜直接监测荧光标记的原肌球蛋白分子与phalloidin稳定的肌动蛋白细丝的协同结合。我们发现原肌凝蛋白分子在单个分子与肌动蛋白随机低亲和力结合后，从多个生长位点组装。随着原肌球蛋白链长度的增加，脱离的可能性降低，从而导致链的进一步生长。原肌球蛋白链延伸与原肌球蛋白浓度呈线性关系，约为100个单体/ μM*s。原肌球蛋白与F -肌动蛋白的随机结合导致不连续的端到端结合，其中链连续性的间隙小于所需的七个连续肌动蛋白单体。直接观察原肌凝蛋白脱离，揭示了肌动蛋白结合的原肌凝蛋白间隙的数量、间隙退火的时间过程以及最终的丝饱和过程。©2015 Wiley期刊公司
Tropomyosin is an elongated α‐helical coiled coil that binds to seven consecutive actin subunits along the long‐pitch helix of actin filaments. Once bound, tropomyosin polymerizes end‐to‐end and both stabilizes F‐actin and regulates access of various actin‐binding proteins including myosin in muscle and nonmuscle cells. Single tropomyosin molecules bind weakly to F‐actin with millimolarKd, whereas the end‐to‐end linked tropomyosin associates with about a 1000‐fold greater affinity. Despite years of study, the assembly mechanism of tropomyosin onto actin filaments remains unclear. In this study, we used total internal reflection fluorescence microscopy to directly monitor the cooperative binding of fluorescently labeled tropomyosin molecules to phalloidin‐stabilized actin filaments. We find that tropomyosin molecules assemble from multiple growth sites after random low‐affinity binding of single molecules to actin. As the length of the tropomyosin chain increases, the probability of detachment decreases, which leads to further chain growth. Tropomyosin chain extension is linearly dependent on the concentration of tropomyosin, occurring at approximately 100 monomers/(μM*s). The random tropomyosin binding to F‐actin leads to discontinuous end‐to‐end association where gaps in the chain continuity smaller than the required seven sequential actin monomers are available. Direct observation of tropomyosin detachment revealed the number of gaps in actin‐bound tropomyosin, the time course of gap annealing, and the eventual filament saturation process. © 2015 Wiley Periodicals, Inc.