Probing structural dynamics of an artificial protein cage using high-speed atomic force microscopy

Area of Science:

• Biophysics
• Structural Biology
• Nanotechnology

Background:

• The ring-shaped protein TRAP (trp-RNA binding attenuation protein) is known to interact with RNA.
• Cysteine-substituted TRAP mutants can self-assemble into higher-order structures.
• Gold nanoparticles can influence protein assembly.

Purpose of the Study:

• To investigate the dynamics of TRAP protein self-assembly into hollow spherical cages induced by gold nanoparticles.
• To visualize the real-time structural changes during TRAP-cage formation and disassembly using high-speed atomic force microscopy (HS-AFM).
• To elucidate the role of gold nanoparticles and disulfide bonds in TRAP-cage assembly and stability.

Main Methods:

• High-speed atomic force microscopy (HS-AFM) for real-time imaging of protein dynamics.
• Utilizing gold nanoparticles as a template for TRAP protein self-assembly.
• Employing reducing agents like dithiothreitol to probe the role of disulfide bonds.

Main Results:

• Direct observation of dynamic TRAP protein aggregation around gold nanoparticles.
• Evidence for gold mediating intermolecular disulfide bond formation during assembly.
• TRAP-cages observed to be composed of multiple, regularly arranged TRAP rings.
• Demonstration that inter-ring disulfide bonds stabilize the TRAP-cage structure.
• Real-time imaging of dramatic TRAP-cage disassembly upon addition of dithiothreitol.

Conclusions:

• HS-AFM provides unprecedented high-resolution, real-time visualization of large protein complex disassembly.
• Gold nanoparticles act as scaffolds, promoting TRAP protein assembly via disulfide bond formation.
• Disulfide bonds are crucial for the structural integrity of the TRAP-cage complex.
• This study offers novel insights into the dynamic assembly and disassembly mechanisms of protein supramolecular structures.