Minakata Shiho, Maeda Kayo, Oda Naoko, Wakabayashi Katsuzo, Nitanai Yasushi, Maéda Yuichiro
ERATO Actin Filament Dynamics Project, JST, Sayo, Hyogo 679-5148, Japan.
Biophys J. 2008 Jul;95(2):710-9. doi: 10.1529/biophysj.107.126144. Epub 2008 Mar 13.
Tropomyosin (Tm) is a two-stranded alpha-helical coiled-coil protein, and when associated with troponin, it is responsible for the actin filament-based regulation of muscle contraction in vertebrate skeletal and cardiac muscles. It is widely believed that Tm adopts a flexible rod-like structure in which the flexibility must play a crucial role in its functions. To obtain more information about the flexibility of Tm, we solved and compared two crystal structures of the identical C-terminal segments, spanning approximately 40% of the entire length. We also compared these structures with our previously reported crystal structure of an almost identical Tm segment in a distinct crystal form. The parameters specifying the local coiled-coil geometry, such as the separation between two helices and the local helical pitch, undulate along the length of Tm in the same way as among the three crystal structures, indicating that these parameters are defined by the amino acid sequence. In the region of increased separation, around Glu-218 and Gln-263, the hydrophobic core is disrupted by three holes. Moreover, for the first time to our knowledge, for Tm, water molecules have been identified in these holes. In some structures, the B-factors are higher around the holes than in the rest of the molecule. The Tm coiled-coil must be destabilized and therefore may be flexible, not only in the alanine clusters but also in the regions of the broken core. A closer look at the local staggering between the two chains and the local bending revealed that the strain accumulates at the alanine cluster and may be relaxed in the broken core region. Moreover, the strain is distributed over a long range, even when a deformation like bending may occur at a limited number of spots. Thus, Tm should not be regarded as a train of short rigid rods connected by flexible linkers, but rather as a seamless rubber rod patched with relatively more flexible regions.
原肌球蛋白（Tm）是一种双链α螺旋卷曲螺旋蛋白，当与肌钙蛋白结合时，它负责脊椎动物骨骼肌和心肌中基于肌动蛋白丝的肌肉收缩调节。人们普遍认为，Tm采用柔性棒状结构，其中的柔性在其功能中必定起着关键作用。为了获取更多关于Tm柔性的信息，我们解析并比较了相同C末端片段的两种晶体结构，该片段跨越了整个长度的约40%。我们还将这些结构与我们之前报道的处于不同晶体形式的几乎相同的Tm片段的晶体结构进行了比较。指定局部卷曲螺旋几何形状的参数，如两条螺旋之间的间距和局部螺旋螺距，在Tm的长度上以与三种晶体结构之间相同的方式波动，这表明这些参数由氨基酸序列定义。在间距增加的区域，围绕Glu - （谷氨酸）218和Gln - （谷氨酰胺）263，疏水核心被三个孔洞破坏。此外，据我们所知，首次在这些孔洞中发现了Tm的水分子。在一些结构中，孔洞周围的B因子比分子的其他部分更高。Tm卷曲螺旋必定不稳定，因此可能是柔性的，不仅在丙氨酸簇中，而且在核心断裂区域也是如此。仔细观察两条链之间的局部交错和局部弯曲发现，应变在丙氨酸簇处积累，并可能在核心断裂区域得到缓解。此外，即使像弯曲这样的变形可能在有限数量的位点发生，应变也会在很长范围内分布。因此，Tm不应被视为由柔性接头连接的一系列短刚性棒，而应被视为用相对更柔性的区域修补的无缝橡胶棒。
