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 和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，探索这些关键动态的方法很少。在这里，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伙伴蛋白质的结合。Central 是一种蛋白质-DNA 偶联策略，它使用核酸外切酶消化和部分重新合成来生成不同长度的 DNA 突出端，并连接到寡核苷酸标记的蛋白质。它提供比现有协议高出 40 倍的耦合产量，并支持新的荧光镊子分析，这需要特别长且坚固的 DNA 手柄。我们通过检测束缚荧光蛋白和与其客户复合的分子伴侣（触发因子）的发射来展示该方法。我们推测我们的策略将成为研究较大生物分子复合物内构象动力学的重要工具。