Mechanical Unfolding of Cardiac Myosin Binding Protein-C by Atomic Force Microscopy
Mechanical Unfolding of Cardiac Myosin Binding Protein-C by Atomic Force Microscopy
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DOI：
10.1016/j.bpj.2011.08.030
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发表时间：
2011-10-19
影响因子：
3.4
通讯作者：
Harris, Samantha P.
中科院分区：
文献类型：
--
作者：
Karsai, Arpad;Kellermayer, Miklos S. Z.;Harris, Samantha P.
心肌肌球蛋白结合蛋白- c （cMyBP-C）是一种厚丝相关蛋白，在心肌肌瘤中起调节和结构作用。它是免疫球蛋白（Ig）超家族的一员，由8个Ig-和3个纤维连接蛋白（FNIII）样结构域组成，以及一个独特的调节序列，称为m结构域，其结构尚不清楚。cMyBP-C c端附近的结构域与肌凝蛋白紧密结合，并介导cMyBP-C与粗（含肌凝蛋白）丝的结合，而n端结构域，包括调节性m结构域，可逆地与肌凝蛋白S2和/或肌动蛋白结合。MyBP-C结合肌凝蛋白和肌动蛋白的能力提高了cMyBP-C在粗丝内交联肌凝蛋白分子和/或将肌凝蛋白和细丝（含肌动蛋白）交联在一起的可能性。在任何一种情况下，cMyBP-C都可能受到机械应变。然而，cMyBP-C的物理性质及其在载荷下的行为是完全未知的。在这里，我们使用原子力显微镜研究了重组杆状病毒表达的cMyBP-C的力学特性，以评估单个cMyBP-C分子在拉伸反应中的稳定性。力扩展曲线显示，在单个Ig和FNIII结构域展开之前，存在较长的可扩展段(s)，其在力谱中表现为锯齿状峰。以500 nm/s的拉伸速率展开Ig/FNIII结构域所需的力从30到150 pN单调增加，表明不同Ig/FNIII结构域之间存在机械层次。使用较小的重组蛋白进行的其他实验表明，调节m结构域缺乏显著的二级或三级结构，可能是cMyBP-C的一个内在紊乱区域。综上所述，这些数据表明cMyBP-C在载荷作用下表现出复杂的力学行为，并且包含具有不同力学性能的多个结构域。
Cardiac myosin-binding protein-C (cMyBP-C) is a thick-filament-associated protein that performs regulatory and structural roles within cardiac sarcomeres. It is a member of the immunoglobulin (Ig) superfamily of proteins consisting of eight Ig- and three fibronectin (FNIII)-like domains, along with a unique regulatory sequence referred to as the M-domain, whose structure is unknown. Domains near the C-terminus of cMyBP-C bind tightly to myosin and mediate the association of cMyBP-C with thick (myosin-containing) filaments, whereas N-terminal domains, including the regulatory M-domain, bind reversibly to myosin S2 and/or actin. The ability of MyBP-C to bind to both myosin and actin raises the possibility that cMyBP-C cross-links myosin molecules within the thick filament and/or cross-links myosin and thin (actin-containing) filaments together. In either scenario, cMyBP-C could be under mechanical strain. However, the physical properties of cMyBP-C and its behavior under load are completely unknown. Here, we investigated the mechanical properties of recombinant baculovirus-expressed cMyBP-C using atomic force microscopy to assess the stability of individual cMyBP-C molecules in response to stretch. Force-extension curves showed the presence of long extensible segment(s) that became stretched before the unfolding of individual Ig and FNIII domains, which were evident as sawtooth peaks in force spectra. The forces required to unfold the Ig/FNIII domains at a stretch rate of 500 nm/s increased monotonically from similar to 30 to similar to 150 pN, suggesting a mechanical hierarchy among the different Ig/FNIII domains. Additional experiments using smaller recombinant proteins showed that the regulatory M-domain lacks significant secondary or tertiary structure and is likely an intrinsically disordered region of cMyBP-C. Together, these data indicate that cMyBP-C exhibits complex mechanical behavior under load and contains multiple domains with distinct mechanical properties.