Wolff-Long V L, Saraswat L D, Lowey S
Rosenstiel Basic Medical Sciences Research Center, Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254-9110.
J Biol Chem. 1993 Nov 5;268(31):23162-7.
Multiple disulfide bonds form in recombinant myosin light chain-2 mutants that contain an engineered cysteine at positions 2, 73, or 94, in addition to the endogenous cysteines at residues 126 and 155 (Saraswat, L.D., and Lowey, S. (1991) J. Biol. Chem. 266, 1977). By replacing one of the native cysteines with an alanine, mutants with a single pair of thiols were created: Cys2/Cys155, Cys2/Cys126, Cys73/Cys155 and Cys94/Cys155. Oxidation of these mutants resulted in a fast migrating band on nonreducing SDS gels, which was attributed to an intramolecular disulfide bond. To determine if disulfide formation could also occur when the light chains (LC) are bound to the myosin heavy chains, LCs were added to myosin which had been depleted of its native LC2 by an immunoadsorbent. When the reconstituted myosin was reacted with 5,5'-dithiobis(2-nitrobenzoic acid) in the absence of divalent cations, intramolecular disulfide bonds formed in the mutant and wild type LCs, but the LCs did not remain bound to the myosin heavy chains. Addition of magnesium ions prevented LC dissociation, but intramolecular disulfide bonds no longer formed. Instead, mutants containing cysteines in the NH2-terminal domain formed intermolecular disulfide bonds between the two heads of myosin. The ability to cross-link the heads demonstrates the existence of close head/head interactions in the myosin molecule, a feature that may be essential for regulation.
在重组肌球蛋白轻链-2突变体中形成了多个二硫键，这些突变体除了在残基126和155处有内源性半胱氨酸外，在第2、73或94位还含有一个工程化的半胱氨酸（萨拉斯瓦特，L.D.，和洛维，S.（1991年）《生物化学杂志》266，1977）。通过用丙氨酸取代其中一个天然半胱氨酸，创建了具有一对巯基的突变体：Cys2/Cys155、Cys2/Cys126、Cys73/Cys155和Cys94/Cys155。这些突变体的氧化导致在非还原SDS凝胶上出现一条快速迁移的条带，这归因于分子内二硫键。为了确定当轻链（LC）与肌球蛋白重链结合时是否也会形成二硫键，将轻链添加到已通过免疫吸附去除其天然LC2的肌球蛋白中。当重组肌球蛋白在没有二价阳离子的情况下与5,5'-二硫代双（2-硝基苯甲酸）反应时，突变体和野生型轻链中形成了分子内二硫键，但轻链不再与肌球蛋白重链结合。添加镁离子可防止轻链解离，但不再形成分子内二硫键。相反，在NH2末端结构域含有半胱氨酸的突变体在肌球蛋白的两个头部之间形成了分子间二硫键。头部交联的能力证明了肌球蛋白分子中存在紧密的头部/头部相互作用，这一特征可能对调节至关重要。
