Valbuena Alejandro, Oroz Javier, Hervás Rubén, Vera Andrés Manuel, Rodríguez David, Menéndez Margarita, Sulkowska Joanna I, Cieplak Marek, Carrión-Vázquez Mariano
Instituto Cajal, Consejo Superior de Investigaciones Científicas and Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, Avenida Doctor Arce 37, E-28002 Madrid, Spain.
Proc Natl Acad Sci U S A. 2009 Aug 18;106(33):13791-6. doi: 10.1073/pnas.0813093106. Epub 2009 Jul 31.
Protein mechanostability is a fundamental biological property that can only be measured by single-molecule manipulation techniques. Such studies have unveiled a variety of highly mechanostable modules (mainly of the Ig-like, beta-sandwich type) in modular proteins subjected to mechanical stress from the cytoskeleton and the metazoan cell-cell interface. Their mechanostability is often attributed to a "mechanical clamp" of secondary structure (a patch of backbone hydrogen bonds) fastening their ends. Here we investigate the nanomechanics of scaffoldins, an important family of scaffolding proteins that assembles a variety of cellulases into the so-called cellulosome, a microbial extracellular nanomachine for cellulose adhesion and degradation. These proteins anchor the microbial cell to cellulose substrates, which makes their connecting region likely to be subjected to mechanical stress. By using single-molecule force spectroscopy based on atomic force microscopy, polyprotein engineering, and computer simulations, here we show that the cohesin I modules from the connecting region of cellulosome scaffoldins are the most robust mechanical proteins studied experimentally or predicted from the entire Protein Data Bank. The mechanostability of the cohesin modules studied correlates well with their mechanical kinetic stability but not with their thermal stability, and it is well predicted by computer simulations, even coarse-grained. This extraordinary mechanical stability is attributed to 2 mechanical clamps in tandem. Our findings provide the current upper limit of protein mechanostability and establish shear mechanical clamps as a general structural/functional motif widespread in proteins putatively subjected to mechanical stress. These data have important implications for the scaffoldin physiology and for protein design in biotechnology and nanotechnology.
蛋白质机械稳定性是一种基本的生物学特性，只能通过单分子操纵技术来测量。此类研究揭示了模块化蛋白质中多种高度机械稳定的模块（主要是免疫球蛋白样的β折叠三明治型），这些蛋白质会受到来自细胞骨架和后生动物细胞间界面的机械应力作用。它们的机械稳定性通常归因于二级结构的“机械夹”（一段主链氢键）固定其末端。在这里，我们研究了支架蛋白的纳米力学，支架蛋白是一类重要的支架蛋白家族，可将多种纤维素酶组装成所谓的纤维小体，这是一种用于纤维素粘附和降解的微生物细胞外纳米机器。这些蛋白质将微生物细胞锚定在纤维素底物上，这使得它们的连接区域可能会受到机械应力。通过基于原子力显微镜的单分子力谱、多蛋白工程和计算机模拟，我们在此表明，来自纤维小体支架蛋白连接区域的黏连蛋白I模块是实验研究的或从整个蛋白质数据库预测的最坚固的机械蛋白。所研究的黏连蛋白模块的机械稳定性与其机械动力学稳定性密切相关，但与其热稳定性无关，并且通过计算机模拟，甚至是粗粒度模拟，都能很好地预测。这种非凡的机械稳定性归因于串联的两个机械夹。我们的发现提供了蛋白质机械稳定性的当前上限，并确立了剪切机械夹作为一种普遍存在于可能受到机械应力作用的蛋白质中的结构/功能基序。这些数据对支架蛋白生理学以及生物技术和纳米技术中的蛋白质设计具有重要意义。
