Probing Structural Dynamics of an Artificial Protein Cage Using High-Speed Atomic Force Microscopy

A cysteine-substituted mutant of the ring-shaped protein TRAP (trp-RNA binding attenuation protein) can be induced to self-assemble into large, monodisperse hollow spherical cages in the presence of 1.4 nm diameter gold nanoparticles. In this study we use high-speed atomic force microscopy (HS-AFM) to probe the dynamics of the structural changes related to TRAP interactions with the gold nanoparticle as well as the disassembly of the cage structure. The dynamic aggregation of TRAP protein in the presence of gold nanoparticles was observed, including oligomeric rearrangements, consistent with a role for gold in mediating intermolecular disulfide bond formation. We were also able to observe that the TRAP-cage is composed of multiple, closely packed TRAP rings in an apparently regular arrangement. A potential role for inter-ring disulfide bonds in forming the TRAP-cage was shown by the fact that ring–ring interactions were reversed upon the addition of reducing agent dithiothreitol. A dramatic disassembly of TRAP-cages was observed using HS-AFM after the addition of dithiothreitol. To the best of our knowledge, this is the first report to show direct high-resolution imaging of the disassembly process of a large protein complex in real time.环形蛋白TRAP（色氨酸-RNA结合衰减蛋白，trp-RNA binding attenuation protein）的半胱氨酸取代突变体，可在直径1.4 nm的金纳米颗粒存在下被诱导自组装形成大尺寸、单分散的空心球形笼状结构。本研究采用高速原子力显微镜（high-speed atomic force microscopy，HS-AFM），探究TRAP与金纳米颗粒相互作用相关的结构动态变化，以及笼状结构的解离过程。研究团队观测到金纳米颗粒存在下TRAP蛋白的动态聚集过程，包括寡聚体重排，这与金介导分子间二硫键形成的作用机制一致。此外，本研究还观测到TRAP笼状结构由多个紧密排布的TRAP环以近似规则的方式组装而成。当向体系中加入还原剂二硫苏糖醇（dithiothreitol）后，环间相互作用被逆转，这表明环间二硫键在TRAP笼状结构的形成过程中可能发挥关键作用。加入二硫苏糖醇后，通过HS-AFM观测到TRAP笼状结构发生了显著解离。据我们所知，本研究首次实现了大型蛋白质复合物解离过程的直接高分辨率实时成像。