Department of Physics, Randall Centre for Cell and Molecular Biophysics and London Centre for Nanotechnology, King's College London, Strand, WC2R 2LS London, United Kingdom.
Single Molecule Mechanobiology Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, London United Kingdom.
Nano Lett. 2022 May 25;22(10):3922-3930. doi: 10.1021/acs.nanolett.2c00043. Epub 2022 May 12.
Non-native disulfide bonds are dynamic covalent bridges that form post-translationally between two cysteines within the same protein (intramolecular) or with a neighboring protein (intermolecular), frequently due to changes in the cellular redox potential. The reversible formation of non-native disulfides is intimately linked to alterations in protein function; while they can provide a mechanism to protect against cysteine overoxidation, they are also involved in the early stages of protein multimerization, a hallmark of several protein aggregation diseases. Yet their identification using current protein chemistry technology remains challenging, mainly because of their fleeting reactivity. Here, we use single-molecule spectroscopy AFM and molecular dynamics simulations to capture both intra- and intermolecular disulfide bonds in γD-crystallin, a cysteine-rich, structural human lens protein involved in age-related eye cataracts. Our approach showcases the power of mechanical force as a conformational probe in dynamically evolving proteins and presents a platform to detect non-native disulfide bridges with single-molecule resolution.
非天然二硫键是一种动态的共价键，在同一个蛋白质内的两个半胱氨酸之间（分子内）或与相邻蛋白质之间（分子间）形成，通常是由于细胞氧化还原电位的变化。非天然二硫键的可逆形成与蛋白质功能的改变密切相关；虽然它们可以提供一种防止半胱氨酸过度氧化的机制，但它们也参与了蛋白质多聚化的早期阶段，这是几种蛋白质聚集疾病的标志。然而，使用当前的蛋白质化学技术来识别它们仍然具有挑战性，主要是因为它们的短暂反应性。在这里，我们使用单分子光谱 AFM 和分子动力学模拟来捕获富含半胱氨酸的结构人晶状体蛋白 γD-晶状体蛋白中的分子内和分子间二硫键，该蛋白与年龄相关性眼白内障有关。我们的方法展示了机械力作为动态演变蛋白质中的构象探针的强大功能，并提供了一个平台，可实现具有单分子分辨率的非天然二硫键桥的检测。
