3 D STRUCTURE OF THE HMM FRAGMENT OF MYOSIN II

在肌球蛋白超家族中，肌球蛋白II是唯一的肌丝形成同种型。 肌球蛋白II是含有两条相同的重链和两对轻链（必需轻链和调节轻链（RLC））的六聚体。 肌球蛋白轻链激酶对RLC的磷酸化是肌球蛋白II中的普遍现象，但其作用因来源而异。 平滑肌和非肌肉肌球蛋白的肌动蛋白激活ATP酶主要由RLC磷酸化调节。 脊椎动物横纹肌肌球蛋白（心肌和骨骼肌）也经历RLC磷酸化，但作用仅是调节ATP酶活性，其主要由细丝上的肌钙蛋白-原肌球蛋白复合物调节。 许多无脊椎动物横纹肌肌球蛋白中的ATP酶活性也是通过肌球蛋白控制的，不是通过轻链磷酸化，而是通过在ELC和RLC之间的界面处与肌球蛋白结合的Ca 2+。肌球蛋白II调节的一个共同特征是两个肌球蛋白头之间的合作相互作用;仅含有一个头的肌球蛋白片段不受调节，并且总是“开启”其肌动蛋白激活的ATP酶活性。 我们最近获得了2-D阵列的平滑肌HMM，一个双头，调节片段的肌球蛋白II，在两个去磷酸化的“关”和磷酸化的“开”状态。 这些阵列适合于通过电子晶体学进行3-成像，并使我们能够首次可视化引起ATP酶抑制的头对头相互作用。 从肌球蛋白II的HMM的2-D结晶促进了以以前无法访问的方式进行调节的研究。 本申请建议将我们对平滑肌肌球蛋白调节的观察扩展到肌球蛋白II的其他亚型，例如扇贝横纹肌肌球蛋白、非肌肉肌球蛋白和脊椎动物横纹肌肌球蛋白，以确定结构相互作用的一般性。

Among the myosin superfamily of motor proteins, myosin II is the sole filament forming isoform. Myosin IIs are hexamers containing two identical heavy chains and two pairs of light chains, the essential light chain and the regulatory light chain (RLC). Phosphorylation of the RLC by myosin light chain kinase is a general phenomenon in myosin IIs but the effect is variable depending on the source. The actin activated ATPase of both smooth and non-muscle myosin is regulated primarily by RLC phosphorylation. Vertebrate striated muscle myosins (cardiac and skeletal) also undergo RLC phosphorylation but the effect is only to modulate the ATPase activity which is regulated primarily by the troponin-tropomyosin complex on the thin filament. ATPase activity in many invertebrate striated muscle myosins is also controlled via myosin, not through light chain phosphorylation, but by Ca2+ binding to myosin at the interface between ELC and RLC. A common feature of myosin II regulation is the cooperative interaction between both myosin heads; myosin fragments that contain only one head are not regulated and are always "on" with respect to their actin activated ATPase activity. We have recently obtained 2-D arrays of smooth muscle HMM, a two-headed, regulated fragment of myosin II, in both the dephosphorylated "off" and phosphorylated "on" states. These arrays are suitable for 3-imaging by electron crystallography and allow us to visualize for the first time, the head to head interactions that give rise to ATPase inhibition. The 2-D crystallization of HMM from myosin II facilitates study of regulation in a way not previously accessible. This application proposes to extend our observations on smooth muscle myosin regulation to other isoforms of myosin II, such as scallop striated, non-muscle and vertebrate striated muscle myosins in order to determine the generality of the structural interactions.