Locking the hydrophobic loop 262-274 to G-actin surface by a disulfide bridge prevents filament formation.
Locking the hydrophobic loop 262-274 to G-actin surface by a disulfide bridge prevents filament formation.
复制标题
通过二硫桥将疏水环262-274锁定至G-肌动蛋白表面可防止细丝形成。
DOI：
10.1021/bi020205f
复制
发表时间：
2002
期刊：
影响因子：
2.9
通讯作者：
Reisler,Emil
中科院分区：
文献类型：
--
作者：
Shvetsov,Alexander;Musib,Runa;Phillips,Martin;Rubenstein,PeterA;Reisler,Emil
F-肌动蛋白结构模型预测了位于亚结构域3和4界面的疏水环262−274对纤维中链间相互作用的重要性。如果这个前提是正确的，那么阻止环构象的改变，即阻止其摆动，就应该中止丝的形成。为了验证这一假设，我们使用定点诱变来产生酵母肌动蛋白三重突变体（LC）2CA（L180 C/L269 C/C374 A）。该突变将两个半胱氨酸残基置于可能使环262−274通过二硫键形成锁定到单体表面的位置。暴露于氧化催化剂的纯化的突变体，导致在SDS PAGE上的肌动蛋白的电泳迁移率增加和两个半胱氨酸的DTNB滴定的损失，与二硫键形成一致。未交联的突变体肌动蛋白的聚合被MgCl 2强烈抑制，但可以恢复到野生型肌动蛋白水平的鬼笔环肽，并通过与野生型肌动蛋白的共聚大大提高。光散射测量显示在相同条件下的交联肌动蛋白的非特异性聚集。电子显微镜证实的情况下，丝和无定形聚集体的存在下，在交联的肌动蛋白样品。在MgCl 2和鬼笔环肽存在下，DTT还原二硫键恢复了正常的肌动蛋白聚合。这些观察结果为疏水环262−274在肌动蛋白聚合成纤维中的关键作用提供了强有力的实验支持。
Models of F-actin structure predict the importance of hydrophobic loop 262−274 at the interface of subdomains 3 and 4 to interstrand interactions in filaments. If this premise is correct, prevention of the loop conformational changeits swinging motionshould abort filament formation. To test this hypothesis, we used site-directed mutagenesis to create yeast actin triple mutant (LC)2CA (L180C/L269C/C374A). This mutation places two cysteine residues in positions potentially enabling the locking of loop 262−274 to the monomer surface via disulfide formation. Exposure of the purified mutant to oxidation catalysts resulted in an increased electrophoretic mobility of actin on SDS PAGE and a loss of two cysteines by DTNB titrations, consistent with disulfide formation. The polymerization of un-cross-linked mutant actin by MgCl2was inhibited strongly but could be restored to wild type actin levels by phalloidin and improved greatly through copolymerization with the wild-type actin. Light scattering measurements revealed nonspecific aggregation of the cross-linked actin under the same conditions. Electron microscopy confirmed the absence of filaments and the presence of amorphous aggregates in the cross-linked actin samples. Reduction of the disulfide bond by DTT restored normal actin polymerization in the presence of MgCl2and phalloidin. These observations provide strong experimental support for a critical role of the hydrophobic loop 262−274 in the polymerization of actin into filaments.