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.

[1]  Toshio Ando,et al.  Filming Biomolecular Processes by High-Speed Atomic Force Microscopy , 2014, Chemical reviews.

[2]  Sara Linse,et al.  Understanding the nanoparticle–protein corona using methods to quantify exchange rates and affinities of proteins for nanoparticles , 2007, Proceedings of the National Academy of Sciences.

[3]  I. Yamashita,et al.  Rounding up: Engineering 12-membered rings from the cyclic 11-mer TRAP. , 2006, Structure.

[4]  Y. Uraoka,et al.  Gold nanoparticle-induced formation of artificial protein capsids. , 2012, Nano letters.

[5]  T. Ando,et al.  A high-speed atomic force microscope for studying biological macromolecules , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[6]  J. Heddle Gold Nanoparticle-Biological Molecule Interactions and Catalysis , 2013 .

[7]  C. Yanofsky,et al.  RNA-based regulation of genes of tryptophan synthesis and degradation, in bacteria. , 2007, RNA.

[8]  Toshio Ando,et al.  High-speed AFM and nano-visualization of biomolecular processes , 2008, Pflügers Archiv - European Journal of Physiology.

[9]  A self-assembled protein nanotube with high aspect ratio. , 2009, Small.

[10]  P. Gollnick Regulation of the Bacillus subtilis trp operon by an RNA‐binding protein , 1994, Molecular microbiology.

[11]  I. Yamashita,et al.  Using the ring-shaped protein TRAP to capture and confine gold nanodots on a surface. , 2007, Small.

[12]  K. Koyasu,et al.  Nonscalable oxidation catalysis of gold clusters. , 2014, Accounts of chemical research.

[13]  Paul Gollnick,et al.  The interaction of RNA with TRAP: the role of triplet repeats and separating spacer nucleotides. , 2004, Journal of molecular biology.

[14]  A. Corma,et al.  Aerobic oxidation of thiols to disulfides by heterogeneous gold catalysts , 2012 .

[15]  Paul Gollnick,et al.  Complexity in regulation of tryptophan biosynthesis in Bacillus subtilis. , 2005, Annual review of genetics.

[16]  A. Antson,et al.  Regulatory features of the trp operon and the crystal structure of the trp RNA-binding attenuation protein from Bacillus stearothermophilus. , 1999, Journal of molecular biology.

[17]  Hiroshi Sano,et al.  Novel Gold Catalysts for the Oxidation of Carbon Monoxide at a Temperature far Below 0 °C , 1987 .

[18]  Brian F. G. Johnson,et al.  Selective oxidation with dioxygen by gold nanoparticle catalysts derived from 55-atom clusters , 2008, Nature.

[19]  Y. Negishi,et al.  Extremely high stability of glutathionate-protected Au25 clusters against core etching. , 2007, Small.