Avellaneda Mario J, Koers Eline J, Minde David P, Sunderlikova Vanda, Tans Sander J
AMOLF, Amsterdam, 1098XG, The Netherlands.
Cambridge Centre for Proteomics, University of Cambridge, Cambridge, CB2 1QR, UK.
Commun Chem. 2020 Feb 12;3(1):20. doi: 10.1038/s42004-020-0267-4.
Many proteins form dynamic complexes with DNA, RNA, and other proteins, which often involves protein conformational changes that are key to function. Yet, methods to probe these critical dynamics are scarce. Here we combine optical tweezers with fluorescence imaging to simultaneously monitor the conformation of individual proteins and their binding to partner proteins. Central is a protein-DNA coupling strategy, which uses exonuclease digestion and partial re-synthesis to generate DNA overhangs of different lengths, and ligation to oligo-labeled proteins. It provides up to 40 times higher coupling yields than existing protocols and enables new fluorescence-tweezers assays, which require particularly long and strong DNA handles. We demonstrate the approach by detecting the emission of a tethered fluorescent protein and of a molecular chaperone (trigger factor) complexed with its client. We conjecture that our strategy will be an important tool to study conformational dynamics within larger biomolecular complexes.
许多蛋白质与DNA、RNA及其他蛋白质形成动态复合物，这通常涉及对功能至关重要的蛋白质构象变化。然而，探测这些关键动力学的方法却很匮乏。在此，我们将光镊与荧光成像相结合，以同时监测单个蛋白质的构象及其与伴侣蛋白的结合。核心是一种蛋白质-DNA偶联策略，该策略利用核酸外切酶消化和部分重新合成来产生不同长度的DNA悬端，并与寡核苷酸标记的蛋白质连接。它比现有方案的偶联产率高出多达40倍，并能实现新的荧光镊检测，而这需要特别长且牢固的DNA柄。我们通过检测 tethered 荧光蛋白以及与其底物复合的分子伴侣（触发因子）的发射来证明该方法。我们推测，我们的策略将成为研究更大生物分子复合物内构象动力学的重要工具。
