Iwamoto Hiroyuki, Oiwa Kazuhiro, Suzuki Takuya, Fujisawa Tetsuro
Life and Environment Division, Japan Synchrotron Radiation Research Institute, Hyogo 679-5198, Japan.
J Mol Biol. 2002 Apr 12;317(5):707-20. doi: 10.1006/jmbi.2002.5449.
The regulatory protein system in the skeletal muscle thin filaments is known to exhibit three discrete states, called "off" or "blocked" (no Ca2+), "on" or "closed" (with Ca2+ alone) and "potentiated" or "open" (with strongly bound myosin head) states. Biochemical studies have shown that only weak interactions with myosin are allowed in the second state. Characterization of each state is often difficult, because the equilibria among these states are readily shifted by experimental conditions. To overcome this problem, we chemically cross-linked the skeletal muscle thin filament in the three states with the zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), in overstretched muscle fibers. The state of the regulatory proteins was monitored by measuring the intensity of the second actin layer-line (2nd LL) reflection in X-ray diffraction patterns. Structurally, the thin filaments cross-linked in the three states exhibited three corresponding discrete levels of 2nd LL intensities, which were not Ca2+-sensitive any more. Functionally, the thin filament cross-linked in the "off-blocked" state inhibited strong interaction with myosin head (subgfragment-1 or S1). The thin filament cross-linked in the "potentiated-open" state allowed strong interaction and full ATPase activity of S1 as described previously. The thin filament cross-linked in the "on-closed" state allowed strong interactions with S1 and actin-activated ATPase without enhancing the 2nd LL to the level of "potentiated-open" state, contrary to the expectations from the biochemical studies. The results demonstrate the potential of EDC as a tool for studying the states of calcium regulation, and the apparent uncoupling between the 2nd LL intensity and the function provides a new insight into the mechanism of thin filament regulation.
已知骨骼肌细肌丝中的调节蛋白系统呈现三种离散状态，即“关闭”或“阻断”状态（无Ca2+）、“开启”或“闭合”状态（仅存在Ca2+）以及“增强”或“开放”状态（存在紧密结合的肌球蛋白头部）。生化研究表明，在第二种状态下，仅允许与肌球蛋白发生微弱相互作用。由于这些状态之间的平衡很容易因实验条件而发生改变，所以对每种状态进行表征往往很困难。为克服这一问题，我们在过度拉伸的肌纤维中，使用零长度交联剂1-乙基-3-(3-二甲基氨基丙基)碳二亚胺（EDC），对处于三种状态的骨骼肌细肌丝进行化学交联。通过测量X射线衍射图谱中第二条肌动蛋白层线（2nd LL）反射的强度，来监测调节蛋白的状态。在结构上，处于三种状态下交联的细肌丝呈现出三种相应的离散水平的2nd LL强度，且不再对Ca2+敏感。在功能上，处于“关闭-阻断”状态下交联的细肌丝抑制了与肌球蛋白头部（亚片段-1或S1）的强烈相互作用。如前所述，处于“增强-开放状态下交联的细肌丝允许S1发生强烈相互作用并具有完全的ATP酶活性。与生化研究的预期相反，处于“开启-闭合”状态下交联的细肌丝允许与S1发生强烈相互作用以及肌动蛋白激活的ATP酶活性，但并未将2nd LL增强至“增强-开放”状态的水平。这些结果证明了EDC作为研究钙调节状态工具的潜力，并且2nd LL强度与功能之间明显的解偶联为细肌丝调节机制提供了新的见解。
