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的金纳米粒子存在下被诱导自组装为大尺寸、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（HS-AFM），对TRAP与金纳米粒子相互作用相关的结构动态变化，以及笼状结构的解聚过程进行了探究。研究观测到金纳米粒子存在下TRAP蛋白的动态聚集现象，包括寡聚体重排，这与金纳米粒子介导分子间二硫键形成的功能一致。此外，研究人员还发现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。环间二硫键在TRAP笼状结构形成中的潜在作用得到验证：当加入还原剂二硫苏糖醇（dithiothreitol）后，环与环之间的相互作用被逆转。在加入二硫苏糖醇后，通过HS-AFM可观测到TRAP笼状结构发生显著解聚。据我们所知，本研究首次实现了大型蛋白复合物解聚过程的直接高分辨率实时成像。