Chakrabarty Tania, Yengo Chris, Baldacchino Corry, Chen Li-Qiong, Sweeney H Lee, Selvin Paul R
Center for Biophysics and Computational Biology and Physics Department, University of Illinois, Urbana, Illinois 61801, USA.
Biochemistry. 2003 Nov 11;42(44):12886-92. doi: 10.1021/bi035144f.
Myosin II, like many molecular motors, is a two-headed dimer held together by a coiled-coil rod. The stability of the (S2) rod has implications for head-head interactions, force generation, and possibly regulation. Whether S2 uncoils has been controversial. To test the stability of S2, we constructed a series of "zippered" dimeric smooth muscle myosin II compounds, containing a high-melting temperature 32-amino acid GCN4 leucine zipper in the S2 rod beginning 0, 1, 2, or 15 heptads from the head-rod junction. We then assessed the ability of these and wild-type myosin to bind strongly via two heads to an actin filament by measuring the fluorescence quenching of pyrene-labeled actin induced by myosin binding. Such two-headed binding is expected to exert a large strain that tends to uncoil S2, and hence provide a robust test of S2 stability. We find that wild-type and zippered heavy meromyosin (HMM) are able to bind by both heads to actin under both nucleotide-free and saturating ADP conditions. In addition, we compared the actin affinity and rates for the 0- and 15-zippered HMMs in the phosphorylated "on" state and found them to be very similar. These results strongly suggest that S2 uncoiling is not necessary for two-headed binding of myosin to actin, presumably due to a compliant point in the myosin head(s). We conclude that S2 likely remains intact during the catalytic cycle.
肌球蛋白II与许多分子马达一样，是一种由卷曲螺旋杆连接在一起的双头二聚体。（S2）杆的稳定性对头对头相互作用、力的产生以及可能的调节都有影响。S2是否解旋一直存在争议。为了测试S2的稳定性，我们构建了一系列“拉链式”二聚体平滑肌肌球蛋白II化合物，在S2杆中从头部-杆连接处开始的第0、1、2或15个七肽处含有一个高熔点的32个氨基酸的GCN4亮氨酸拉链。然后，我们通过测量肌球蛋白结合诱导的芘标记肌动蛋白的荧光猝灭，评估这些化合物和野生型肌球蛋白通过两个头部与肌动蛋白丝强烈结合的能力。这种双头结合预计会产生很大的应变，倾向于使S2解旋，因此能对S2的稳定性进行有力测试。我们发现野生型和拉链式重酶解肌球蛋白（HMM）在无核苷酸和饱和ADP条件下都能通过两个头部与肌动蛋白结合。此外，我们比较了磷酸化“开启”状态下0拉链和15拉链HMM的肌动蛋白亲和力和速率，发现它们非常相似。这些结果强烈表明，肌球蛋白与肌动蛋白的双头结合并不需要S2解旋，推测是由于肌球蛋白头部存在一个柔性点。我们得出结论，在催化循环过程中S2可能保持完整。
