Data supporting Cell-free expression with a quartz crystal microbalance enables rapid, dynamic, and label-free characterization of membrane-interacting proteins

Integral and interacting membrane proteins (IIMPs) constitute a vast family of biomolecules that perform essential functions in all forms of life. However, characterizing their interactions with lipid bilayers remains limited due to challenges in purifying and reconstituting IIMPs in vitro or labeling IIMPs without disrupting their function in vivo. Here, we report cell-free transcription-translation in a quartz crystal microbalance with dissipation (TXTL-QCMD) to dynamically characterize interactions between diverse IIMPs and membranes without protein purification or labeling. As part of TXTL-QCMD, IIMPs are synthesized using cell-free transcription-translation (TXTL), and their interactions with supported lipid bilayers are measured using a quartz crystal microbalance with dissipation (QCMD). TXTL-QCMD reconstitutes known IIMP-membrane dependencies, including specific association with prokaryotic or eukaryotic membranes, and the multiple-IIMP dynamical pattern-forming association of the E. coli division-coordinating proteins MinCDE. Applying TXTL-QCMD to the recently discovered Zorya anti-phage system that is unamenable to labeling, we discovered that ZorA and ZorB integrate within the lipids found at the poles of bacteria while ZorE diffuses freely on the non-pole membrane. These efforts establish the potential of TXTL-QCMD to broadly characterize the large diversity of IIMPs.

整合型相互作用膜蛋白（integral and interacting membrane proteins, IIMPs）是一类分布广泛的生物分子家族，在所有生命形式中均发挥着不可或缺的重要功能。然而，由于体外纯化与重构IIMPs，或是在不破坏其体内功能的前提下对IIMPs进行标记均存在诸多技术难题，目前对其与脂质双层的相互作用进行表征的研究仍较为有限。本研究报道了一种将无细胞转录翻译（cell-free transcription-translation, TXTL）体系与耗散型石英晶体微天平（quartz crystal microbalance with dissipation, QCMD）平台相结合的技术（命名为TXTL-QCMD），可在无需蛋白质纯化或标记的条件下，动态表征多种IIMPs与膜结构之间的相互作用。在TXTL-QCMD的技术流程中，IIMPs通过无细胞转录翻译体系完成合成，并通过耗散型石英晶体微天平对其与支撑脂质双层的相互作用进行检测。该技术可重现已知的IIMP与膜的依赖关系，包括其与原核或真核膜的特异性结合，以及大肠杆菌分裂协调蛋白MinCDE所形成的多IIMP动态模式结合行为。将TXTL-QCMD应用于此前发现的无法进行标记的Zorya抗噬菌体系统时，我们发现ZorA与ZorB可整合至细菌极区的脂质中，而ZorE则可在细菌非极区膜上自由扩散。本研究证实了TXTL-QCMD技术具备广泛表征种类繁多的IIMPs的应用潜力。