Messer N, Kendrick-Jones J
MRC Laboratory of Molecular Biology, Cambridge, U.K.
J Mol Biol. 1991 Apr 20;218(4):825-35. doi: 10.1016/0022-2836(91)90270-g.
The regulatory light chains (RLCs) located on the myosin head, regulate the interaction of myosin with actin in response to either Ca2+ or phosphorylation signals. The RLCs belong to a family of calcium binding proteins and are composed of four "EF hand" ancestral calcium binding motifs (numbered I to IV). To determine the role of the first EF hand (EF hand I) in the regulatory process, chimaeric light chains were constructed by protein engineering, by switching this region between smooth muscle and skeletal muscle myosin RLCs. For example, chimaera G(I)S consisted of EF hand I of the smooth muscle (gizzard) RLC and EF hands II to IV of the skeletal muscle RLC, whereas chimaera S(I)G consisted of EF hand I of the skeletal muscle RLC and EF hands II to IV of the smooth muscle RLC. The chimaeric RLCs were expressed in Escherichia coli using the pLcII expression system, and after isolation and purification their regulatory properties were compared with those of wild-type smooth and skeletal muscle myosin RLCs. The chimaeric RLCs bound to the myosin heads in scallop striated muscle myofibrils from which the endogenous RLCs had been removed ("desensitized" myofibrils) with similar affinities to those of the wild-type smooth and skeletal muscle RLCs. Both chimaeric RLCs were able to regulate the actin-activated Mg(2+)-ATPase activity of scallop myosin: G(I)S inhibited the ATPase in the presence and absence of Ca2+, like the wild-type skeletal muscle RLC, while S(I)G inhibited the myosin ATPase in the absence of Ca2+, and this inhibition was relieved on Ca2+ addition, in the same way as the wild-type smooth muscle RLC. Thus the type of regulation that the RLCs confer on the myosin is determined by the source of EF hands II to IV rather than that of EF hand I.
位于肌球蛋白头部的调节性轻链（RLCs），响应Ca2+或磷酸化信号来调节肌球蛋白与肌动蛋白的相互作用。RLCs属于钙结合蛋白家族，由四个“EF手型”祖先钙结合基序（编号为I至IV）组成。为了确定第一个EF手型（EF手型I）在调节过程中的作用，通过蛋白质工程构建了嵌合轻链，方法是在平滑肌和骨骼肌肌球蛋白RLCs之间切换该区域。例如，嵌合体G(I)S由平滑肌（砂囊）RLC的EF手型I和骨骼肌RLC的EF手型II至IV组成，而嵌合体S(I)G由骨骼肌RLC的EF手型I和平滑肌RLC的EF手型II至IV组成。使用pLcII表达系统在大肠杆菌中表达嵌合RLCs，分离和纯化后，将它们的调节特性与野生型平滑肌和骨骼肌肌球蛋白RLCs的调节特性进行比较。嵌合RLCs以与野生型平滑肌和骨骼肌RLCs相似的亲和力与扇贝横纹肌肌原纤维中的肌球蛋白头部结合，这些肌原纤维中的内源性RLCs已被去除（“脱敏”肌原纤维）。两种嵌合RLCs都能够调节扇贝肌球蛋白的肌动蛋白激活的Mg(2+)-ATPase活性：G(I)S在有和没有Ca2+的情况下都抑制ATPase，就像野生型骨骼肌RLC一样，而S(I)G在没有Ca2+的情况下抑制肌球蛋白ATPase，并且在添加Ca2+时这种抑制作用解除，与野生型平滑肌RLC的方式相同。因此，RLCs赋予肌球蛋白的调节类型是由EF手型II至IV的来源而不是EF手型I的来源决定的。
