Shvetsov Alexander, Musib Runa, Phillips Martin, Rubenstein Peter A, Reisler Emil
Department of Chemistry and Biochemistry and Molecular Biology Institute, University of California, Los Angeles, California 90095, USA.
Biochemistry. 2002 Sep 3;41(35):10787-93. doi: 10.1021/bi020205f.
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 change--its swinging motion--should 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 MgCl2 was 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 MgCl2 and phalloidin. These observations provide strong experimental support for a critical role of the hydrophobic loop 262-274 in the polymerization of actin into filaments.
F-肌动蛋白结构模型预测，在亚结构域3和4的界面处，疏水环262 - 274对细丝中的链间相互作用很重要。如果这个前提是正确的，那么防止环构象变化——其摆动运动——应该会阻止细丝形成。为了验证这一假设，我们使用定点诱变技术创建了酵母肌动蛋白三重突变体(LC)2CA（L180C/L269C/C374A）。这种突变在两个位置引入了半胱氨酸残基，这可能使262 - 274环通过二硫键形成锁定在单体表面。将纯化的突变体暴露于氧化催化剂下，导致肌动蛋白在SDS-PAGE上的电泳迁移率增加，并且通过DTNB滴定法检测到两个半胱氨酸的损失，这与二硫键的形成一致。MgCl2介导的未交联突变体肌动蛋白的聚合受到强烈抑制，但可以通过鬼笔环肽恢复到野生型肌动蛋白水平，并且通过与野生型肌动蛋白共聚可以大大改善。光散射测量显示在相同条件下交联肌动蛋白存在非特异性聚集。电子显微镜证实交联肌动蛋白样品中没有细丝，只有无定形聚集体。在MgCl2和鬼笔环肽存在的情况下，用DTT还原二硫键可恢复正常的肌动蛋白聚合。这些观察结果为疏水环262 - 274在肌动蛋白聚合成细丝过程中的关键作用提供了有力的实验支持。
