Hsp27-actin interaction.
Hsp27-actin interaction.
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DOI：
10.1155/2011/901572
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发表时间：
2011
影响因子：
3
通讯作者：
Graceffa P
中科院分区：
文献类型：
--
作者：
Graceffa P
据报道，Hsp27寡聚体与F-肌动蛋白以带刺末端封端蛋白的形式相互作用。本研究利用连接到HSP27半胱氨酸-137上的荧光探针测定了相互作用的结合强度和化学计量比。与F-肌动蛋白相互作用后，连接在HSP27上的阿克洛坦的荧光强度增加了4-5倍。与F-肌动蛋白的荧光滴定得到一个弱结合常数(K D APP=5.3 μM)，其肌动蛋白/HSP2 7化学计量比介于 1和6之间。该化学计量比与F-肌动蛋白末端封端蛋白不一致。连接到HSP27上的芘显示出很大的准分子荧光，这与已知的HSP27齐聚物中半胱氨酸-137‘S的存在是一致的。当与F-肌动蛋白相互作用时，Py-Hsp27的准分子荧光大部分丢失，这表明Hsp27与F-肌动蛋白以单体相互作用，这与acryloan-Hsp27的结果一致。F-肌动蛋白-Hsp27的电子显微镜图像显示，Hsp27不是一个很强的G-肌动蛋白隔离物。因此，在体外，Hsp27是一种弱的F-肌动蛋白侧边结合蛋白。
Hsp27 oligomer is reported to interact with F-actin as a barbed-end-capping protein. The present study determined the binding strength and stoichiometry of the interaction using fluorescence of probes attached to Hsp27 cysteine-137. The fluorescence of acrylodan attached to Hsp27 increased 4-5-fold upon interaction with F-actin. Titration of the fluorescence with F-actin yielded a weak binding constant (K D app = 5.3 μM) with an actin/Hsp27 stoichiometry between < 1 and 6. This stoichiometry is inconsistent with an F-actin end-capping protein. Pyrene attached to Hsp27 exhibited a large excimer fluorescence, in agreement with the known proximity of the cysteine-137's in the Hsp27 oligomer. Upon interaction with F-actin the pyrene-Hsp27 excimer fluorescence was largely lost, suggesting that Hsp27 interacts with F-actin as a monomer, consistent with the acrylodan-Hsp27 results. EM images of F-actin-Hsp27 demonstrated that Hsp27 is not a strong G-actin sequester. Thus, Hsp27, in vitro, is a weak F-actin side-binding protein.