Ueno H, Harrington W F
J Mol Biol. 1984 Feb 15;173(1):35-61. doi: 10.1016/0022-2836(84)90402-9.
The temperature-dependence of local melting within the alpha-helical, coiled-coil structure of rabbit myosin rod has been investigated by following changes in the rate constants of proteolytic digestion. The kinetics of fragmentation of the rod by three different enzymes (alpha-chymotrypsin, trypsin and papain) over the temperature range 5 to 40 degrees C (pH 7, I = 0.5) has been monitored by electrophoresis of the digestion products on sodium dodecyl sulfate/polyacrylamide gels. All rate constants were corrected for the intrinsic temperature-dependence of the enzyme by comparison with model substrates. Results from the three enzyme-probes are similar in showing that local melting within the rod occurs in two distinct stages. At temperatures between 5 and 25 degrees C, melting is confined to a restricted segment of the rod structure near the light meromyosin/heavy meromyosin junction. At temperatures between 25 and 40 degrees C, a wider segment of the rod lysing between the junction and the short subfragment-2 segment (the hinge domain) appears to be melting, judging from the broad spectrum of cleavage sites observed in this region. Results are compared with those from other physicochemical methods that measure the hinging or opening of the coiled-coil structure of the rod.
通过跟踪蛋白水解消化速率常数的变化，研究了兔肌球蛋白杆状结构中α-螺旋卷曲螺旋结构内局部解链的温度依赖性。在5至40摄氏度（pH 7，离子强度I = 0.5）的温度范围内，用三种不同的酶（α-胰凝乳蛋白酶、胰蛋白酶和木瓜蛋白酶）使杆状结构片段化的动力学过程，通过在十二烷基硫酸钠/聚丙烯酰胺凝胶上对消化产物进行电泳来监测。通过与模型底物比较，对所有速率常数进行了酶固有温度依赖性的校正。三种酶探针的结果相似，表明杆状结构内的局部解链分两个不同阶段发生。在5至25摄氏度之间的温度下，解链局限于杆状结构靠近轻酶解肌球蛋白/重酶解肌球蛋白连接处的一个受限片段。在25至40摄氏度之间的温度下，从该区域观察到的广泛切割位点判断，杆状结构在连接处和短的亚片段-2片段（铰链区）之间裂解的更宽片段似乎正在解链。将结果与其他测量杆状结构卷曲螺旋结构铰链或打开情况的物理化学方法的结果进行了比较。
