Manteca Aitor, Alonso-Caballero Álvaro, Fertin Marie, Poly Simon, De Sancho David, Perez-Jimenez Raul
From the Nanobiomechanics Laboratory, CIC nanoGUNE, 20018 Donostia-San Sebastián, Spain.
the Interfaculty Institute of Biochemistry, University of Tübingen, 72076 Tübingen, Germany, and.
J Biol Chem. 2017 Aug 11;292(32):13374-13380. doi: 10.1074/jbc.M117.784934. Epub 2017 Jun 22.
Disulfide bonds play a crucial role in proteins, modulating their stability and constraining their conformational dynamics. A particularly important case is that of proteins that need to withstand forces arising from their normal biological function and that are often disulfide bonded. However, the influence of disulfides on the overall mechanical stability of proteins is poorly understood. Here, we used single-molecule force spectroscopy (smFS) to study the role of disulfide bonds in different mechanical proteins in terms of their unfolding forces. For this purpose, we chose the pilus protein FimG from Gram-negative bacteria and a disulfide-bonded variant of the I91 human cardiac titin polyprotein. Our results show that disulfide bonds can alter the mechanical stability of proteins in different ways depending on the properties of the system. Specifically, disulfide-bonded FimG undergoes a 30% increase in its mechanical stability compared with its reduced counterpart, whereas the unfolding force of I91 domains experiences a decrease of 15% relative to the WT form. Using a coarse-grained simulation model, we rationalized that the increase in mechanical stability of FimG is due to a shift in the mechanical unfolding pathway. The simple topology-based explanation suggests a neutral effect in the case of titin. In summary, our results indicate that disulfide bonds in proteins act in a context-dependent manner rather than simply as mechanical lockers, underscoring the importance of considering disulfide bonds both computationally and experimentally when studying the mechanical properties of proteins.
二硫键在蛋白质中起着至关重要的作用，调节其稳定性并限制其构象动力学。一个特别重要的情况是那些需要承受正常生物学功能产生的力且通常通过二硫键结合的蛋白质。然而，二硫键对蛋白质整体机械稳定性的影响却知之甚少。在这里，我们使用单分子力谱（smFS）从展开力的角度研究二硫键在不同机械蛋白中的作用。为此，我们选择了革兰氏阴性菌的菌毛蛋白FimG和人心脏肌联蛋白多蛋白I91的二硫键结合变体。我们的结果表明，二硫键可根据系统特性以不同方式改变蛋白质的机械稳定性。具体而言，与还原形式相比，二硫键结合的FimG的机械稳定性提高了30%，而I91结构域的展开力相对于野生型形式降低了15%。使用粗粒度模拟模型，我们推断FimG机械稳定性的提高是由于机械展开途径的转变。基于简单拓扑结构的解释表明肌联蛋白的情况是中性的。总之，我们的结果表明蛋白质中的二硫键以依赖于上下文的方式起作用，而不是简单地作为机械锁定物，这突出了在研究蛋白质机械特性时在计算和实验中考虑二硫键的重要性。
