title: Probing structural dynamics of an artificial protein cage using high-speed atomic force microscopy.
abstract: 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.
authors.affiliation: Heddle Initiative Research Unit, RIKEN, Wako, Saitama 351-0198, Japan.
authors.forename: Motonori
authors.initials: M
authors.lastname: Imamura
authors.forename: Takayuki
authors.initials: T
authors.lastname: Uchihashi
authors.forename: Toshio
authors.initials: T
authors.lastname: Ando
authors.forename: Annika
authors.initials: A
authors.lastname: Leifert
authors.forename: Ulrich
authors.initials: U
authors.lastname: Simon
authors.forename: Ali D
authors.initials: AD
authors.lastname: Malay
authors.forename: Jonathan G
authors.initials: JG
authors.lastname: Heddle
mesh_terms: D018625:Microscopy, Atomic Force / Q000379:methods*; D015335:Molecular Probes; D011506:Proteins / Q000737:chemistry*
chemical_list: D015335:Molecular Probes; D011506:Proteins
keywords: Protein cage; disulfide bond; gold nanoparticle; high-speed atomic force microscopy; protein disassembly; protein folding