Suzuki Y, Yasunaga T, Ohkura R, Wakabayashi T, Sutoh K
Department of Life Sciences, School of Arts and Sciences, University of Tokyo, Japan.
Nature. 1998 Nov 26;396(6709):380-3. doi: 10.1038/24640.
In muscle, the myosin head ('crossbridge') performs the 'working stroke', in which ATP is hydrolysed to generate the sliding of actin and myosin filaments. The myosin head consists of a globular motor domain and a long lever-arm domain. The 'lever-arm hypothesis' predicts that during the working stroke, the lever-arm domain tilts against the motor domain, which is bound to actin in a fixed orientation. To detect this working stroke in operation, we constructed fusion proteins by connecting Aequorea victoria green fluorescent protein and blue fluorescent protein to the amino and carboxyl termini of the motor domain of myosin II of Dictyostelium discoideum, a soil amoeba, and measured the fluorescence resonance energy transfer between the two fluorescent proteins. We show here that the carboxy-terminal fluorophore swings at the isomerization step of the ATP hydrolysis cycle, and then swings back at the subsequent step in which inorganic phosphate is released, thereby mimicking the swing of the lever arm. The swing at the phosphate-release step may correspond to the working stroke, and the swing at the isomerization step to the recovery stroke.
在肌肉中，肌球蛋白头部（“横桥”）执行“工作冲程”，其中ATP被水解以产生肌动蛋白和肌球蛋白丝的滑动。肌球蛋白头部由一个球状运动结构域和一个长的杠杆臂结构域组成。“杠杆臂假说”预测，在工作冲程期间，杠杆臂结构域相对于运动结构域倾斜，运动结构域以固定方向与肌动蛋白结合。为了检测正在进行的这种工作冲程，我们通过将维多利亚多管发光水母绿色荧光蛋白和蓝色荧光蛋白连接到一种土壤变形虫——盘基网柄菌肌球蛋白II运动结构域的氨基和羧基末端来构建融合蛋白，并测量了两种荧光蛋白之间的荧光共振能量转移。我们在此表明，羧基末端荧光团在ATP水解循环的异构化步骤摆动，然后在随后释放无机磷酸盐的步骤中又摆动回来，从而模拟了杠杆臂的摆动。磷酸盐释放步骤的摆动可能对应于工作冲程，而异构化步骤的摆动对应于恢复冲程。
