Schoenauer Roman, Bertoncini Patricia, Machaidze Gia, Aebi Ueli, Perriard Jean-Claude, Hegner Martin, Agarkova Irina
Institute of Cell Biology, ETH Zürich-Hönggerberg, CH-8093 Zürich, Switzerland.
J Mol Biol. 2005 Jun 3;349(2):367-79. doi: 10.1016/j.jmb.2005.03.055. Epub 2005 Apr 7.
The M-band is a transverse structure in the center of the sarcomere, which is thought to stabilize the thick filament lattice. It was shown recently that the constitutive vertebrate M-band component myomesin can form antiparallel dimers, which might cross-link the neighboring thick filaments. Myomesin consists mainly of immunoglobulin-like (Ig) and fibronectin type III (Fn) domains, while several muscle types express the EH-myomesin splice isoform, generated by the inclusion of the unique EH-segment of about 100 amino acid residues (aa) in the center of the molecule. Here we use atomic force microscopy (AFM), transmission electron microscopy (TEM) and circular dichroism (CD) spectroscopy for the biophysical characterization of myomesin. The AFM identifies the "mechanical fingerprints" of the modules constituting the myomesin molecule. Stretching of homomeric polyproteins, constructed of Ig and Fn domains of human myomesin, produces a typical saw-tooth pattern in the force-extension curve. The domains readily refold after relaxation. In contrast, stretching of a heterogeneous polyprotein, containing several repeats of the My6-EH fragment reveals a long initial plateau corresponding to the sum of EH-segment contour lengths, followed by several My6 unfolding peaks. According to this, the EH-segment is characterized as an entropic chain with a persistence length of about 0.3nm. In TEM pictures, the EH-domain appears as a gap in the molecule, indicating a random coil conformation similar to the PEVK region of titin. CD spectroscopy measurements support this result, demonstrating a mostly non-folded conformation for the EH-segment. We suggest that similarly to titin, myomesin is a molecular spring, whose elasticity is modulated by alternative splicing. The Ig and Fn domains might function as reversible "shock absorbers" by sequential unfolding in the case of extremely high or long sustained stretching forces. These complex visco-elastic properties of myomesin might be crucial for the stability of the sarcomere.
M带是肌节中心的一种横向结构，被认为可稳定粗肌丝晶格。最近有研究表明，组成脊椎动物M带的肌间蛋白可形成反平行二聚体，可能会交联相邻的粗肌丝。肌间蛋白主要由免疫球蛋白样（Ig）和纤连蛋白III型（Fn）结构域组成，而几种肌肉类型表达EH-肌间蛋白剪接异构体，它是通过在分子中心包含约100个氨基酸残基（aa）的独特EH片段而产生的。在此，我们使用原子力显微镜（AFM）、透射电子显微镜（TEM）和圆二色性（CD）光谱对肌间蛋白进行生物物理表征。AFM可识别构成肌间蛋白分子的模块的“机械指纹”。由人肌间蛋白的Ig和Fn结构域构建的同聚多蛋白的拉伸，在力-伸长曲线中产生典型的锯齿状模式。结构域在松弛后很容易重新折叠。相比之下，包含多个My6-EH片段重复序列的异质多蛋白的拉伸显示出一个长的初始平台期，对应于EH片段轮廓长度的总和，随后是几个My6解折叠峰。据此，EH片段被表征为具有约0.3nm持久长度的熵链。在TEM图像中，EH结构域在分子中表现为一个间隙，表明其构象类似于肌联蛋白的PEVK区域的无规卷曲。CD光谱测量支持这一结果，表明EH片段大多处于未折叠构象。我们认为，与肌联蛋白类似，肌间蛋白是一种分子弹簧，其弹性由可变剪接调节。在极高或长时间持续拉伸力的情况下，Ig和Fn结构域可能通过顺序解折叠作为可逆的“减震器”发挥作用。肌间蛋白的这些复杂粘弹性特性可能对肌节的稳定性至关重要。
