Smooth muscle myosin mutants containing a single tryptophan reveal molecular interactions at the actin-binding interface.
Smooth muscle myosin mutants containing a single tryptophan reveal molecular interactions at the actin-binding interface.
复制标题
含有单个色氨酸的平滑肌肌球蛋白突变体揭示了肌动蛋白结合界面上的分子相互作用。
DOI：
10.1073/pnas.95.22.12944
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发表时间：
1998
影响因子：
11.1
通讯作者：
Berger,CL
中科院分区：
文献类型：
--
作者：
Yengo,CM;Fagnant,PM;Chrin,L;Rovner,AS;Berger,CL
阐明的环状肌动球蛋白相互作用的分子细节需要能够检查肌动蛋白结合界面的肌球蛋白在特定位点的结构变化。为了动态地研究这些变化，我们表达了两个突变体的截短片段的鸡砂囊平滑肌肌球蛋白，其中包括电机域和必需轻链（MDE）。这些突变体被改造为在（Trp-546）或附近（Trp-625）的推定肌动蛋白结合界面含有一个单一的色氨酸。546-和625-MDE均表现出与野生型MDE相似的肌动蛋白激活的ATP酶和肌动蛋白结合活性。546-和625-MDE的荧光发射光谱和丙烯酰胺淬灭表明，在存在和不存在ATP的情况下，Trp-546几乎完全暴露于溶剂，而Trp-625的暴露量小于50%，与现有的晶体结构数据一致。与肌动蛋白结合的625-MDE的光谱与未结合的光谱非常相似，这表明，尽管Trp-625位于肌球蛋白的50/20-kDa环和50-kDa裂缝附近，但其构象在肌动蛋白结合时不会改变。然而，在肌动蛋白存在下观察到的546-MDE的峰值发射波长蓝移10 nm表明，色氨酸-546，位于较低的50-kDa的肌球蛋白亚结构域的A-位点，存在于一个更深埋的环境中，并可能直接与肌动蛋白的严格acto-S1复合物相互作用。Trp-546光谱的这种变化构成了肌球蛋白和肌动蛋白A位点残基之间特异性分子相互作用的直接证据。
Elucidation of the molecular details of the cyclic actomyosin interaction requires the ability to examine structural changes at specific sites in the actin-binding interface of myosin. To study these changes dynamically, we have expressed two mutants of a truncated fragment of chicken gizzard smooth muscle myosin, which includes the motor domain and essential light chain (MDE). These mutants were engineered to contain a single tryptophan at (Trp-546) or near (Trp-625) the putative actin-binding interface. Both 546- and 625-MDE exhibited actin-activated ATPase and actin-binding activities similar to wild-type MDE. Fluorescence emission spectra and acrylamide quenching of 546- and 625-MDE suggest that Trp-546 is nearly fully exposed to solvent and Trp-625 is less than 50% exposed in the presence and absence of ATP, in good agreement with the available crystal structure data. The spectrum of 625-MDE bound to actin was quite similar to the unbound spectrum indicating that, although Trp-625 is located near the 50/20-kDa loop and the 50-kDa cleft of myosin, its conformation does not change upon actin binding. However, a 10-nm blue shift in the peak emission wavelength of 546-MDE observed in the presence of actin indicates that Trp-546, located in the A-site of the lower 50-kDa subdomain of myosin, exists in a more buried environment and may directly interact with actin in the rigor acto-S1 complex. This change in the spectrum of Trp-546 constitutes direct evidence for a specific molecular interaction between residues in the A-site of myosin and actin.