Wolff-Long V L, Tao T, Lowey S
Rosenstiel Basic Medical Sciences Research Center, Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254, USA.
J Biol Chem. 1995 Dec 29;270(52):31111-8. doi: 10.1074/jbc.270.52.31111.
Resonance energy transfer was used to measure the distances between pairs of cysteines, Cys2 and Cys155 and Cys73 and Cys155, in recombinant chicken skeletal myosin regulatory light chains in the free and bound states. The fluorescent and nonfluorescent probes N-iodoacetyl-N'-(5-sulfo-1-naphthyl) ethylenediamine and N-(4-dimethylamino-3,5-dinitrophenyl)maleimide were used as the donor and the acceptor, respectively. The distance between Cys2 and Cys155 was measured to be 35 and 30 A in the absence and presence of myosin heavy chain, respectively, suggesting a slightly more compact structure for the light chain in the bound state. The distance between Cys73 and Cys155 measured in a similar manner was 31 and 30 A in the free and bound states, respectively; this latter value is in good agreement with that derived from crystallographic structures. For heavy chain-bound light chains, no measurable distance changes were detected with the binding of ATP or actin. These results show that no gross structural changes occur within the regulatory light chain during the contraction cycle, but that resonance energy transfer between other sites could be used to monitor potential changes in the myosin head upon the binding of nucleotides and actin.
共振能量转移被用于测量重组鸡骨骼肌肌球蛋白调节轻链在游离态和结合态时，半胱氨酸对（Cys2与Cys155以及Cys73与Cys155）之间的距离。荧光探针N-碘乙酰基-N'-(5-磺基-1-萘基)乙二胺和非荧光探针N-(4-二甲基氨基-3,5-二硝基苯基)马来酰亚胺分别用作供体和受体。在不存在和存在肌球蛋白重链的情况下，测得Cys2与Cys155之间的距离分别为35 Å和30 Å，这表明结合态的轻链结构稍紧凑一些。以类似方式测得的Cys73与Cys155在游离态和结合态之间的距离分别为31 Å和30 Å；后一个值与晶体结构得出的值高度吻合。对于重链结合的轻链，在结合ATP或肌动蛋白时未检测到可测量的距离变化。这些结果表明，在收缩周期中调节轻链内未发生明显的结构变化，但其他位点之间的共振能量转移可用于监测核苷酸和肌动蛋白结合后肌球蛋白头部的潜在变化。
