Kellermayer M S, Granzier H L
Department of Veterinary Comparative Anatomy, Pharmacology and Physiology, Washington State University, Pullman 99164-6520, USA.
Biochem Biophys Res Commun. 1996 Apr 25;221(3):491-7. doi: 10.1006/bbrc.1996.0624.
Titin (also known as connection) is a giant filamentous protein that spans the distance between the Z- and M-lines of the vertebrate muscle sarcomere. Several indirect observations have implicated titin as playing a fundamental role in the generation of passive force of muscle, driven by titin's elastic properties. A direct observation of the mechanical properties of titin, however, has not been demonstrated. Here we have used the recently shown strong actin-binding property of titin to indirectly visualize and manipulate single molecules of titin. Titin molecules were immobilized on a microscope coverslip by attaching them to anti-titin antibody. The titin molecules were detected by attaching fluorescent actin filaments to them. The titin molecules were subsequently stretched by manipulating the free end of the attached actin filaments with a glass microneedle. Titin is shown here to possess a high degree of torsional and longitudinal flexibility. The molecule can be repetitively stretched at least fourfold, followed by recoil. Titin's unloaded elastic recoil proceeded in two stages: an initial rapid process (15 ms time constant) was followed by a slower one (400 ms time constant). The force necessary to fully extend titin--estimated by observing the breakage of the titin-bound actin filaments--may reach above approximately 100 pN (longitudinal tensile strength of actin). Attachment of fluorescent actin filaments to titin provides a useful tool to further probe titin's molecular properties.
肌联蛋白（也称为连接蛋白）是一种巨大的丝状蛋白，它跨越脊椎动物肌肉肌节的Z线和M线之间的距离。一些间接观察表明，肌联蛋白凭借其弹性特性，在肌肉被动力的产生中发挥着重要作用。然而，尚未直接观察到肌联蛋白的力学特性。在此，我们利用最近发现的肌联蛋白与肌动蛋白的强结合特性，间接可视化并操控单个肌联蛋白分子。通过将肌联蛋白分子与抗肌联蛋白抗体相连，将其固定在显微镜盖玻片上。通过将荧光肌动蛋白丝附着于肌联蛋白分子来检测它们。随后，用玻璃微针操控附着的肌动蛋白丝的自由端，从而拉伸肌联蛋白分子。结果表明，肌联蛋白具有高度的扭转和纵向柔韧性。该分子可以反复拉伸至少四倍，然后回缩。肌联蛋白的无负载弹性回缩分两个阶段进行：初始的快速过程（时间常数为15毫秒）之后是较慢的过程（时间常数为400毫秒）。通过观察与肌联蛋白结合的肌动蛋白丝的断裂来估计，使肌联蛋白完全伸展所需的力可能达到约100皮牛以上（肌动蛋白的纵向拉伸强度）。将荧光肌动蛋白丝附着于肌联蛋白为进一步探究肌联蛋白的分子特性提供了一种有用的工具。
