Cell-Free Nonequilibrium Assembly for Hierarchical Protein/Peptide Nanopillars

Area of Science:

• Biomaterials Science
• Nanotechnology
• Cellular Biology

Background:

• Cells possess complex protein nanostructures that are difficult to replicate outside of cellular environments.
• Vertical fibronectin pillars in embryos serve as a model for intricate, naturally occurring protein assemblies.
• Existing methods face challenges in recreating the directional growth and composition of these cellular structures.

Purpose of the Study:

• To develop a cell-free method for creating vertical fibronectin pillar mimics.
• To investigate the use of enzyme-responsive phosphopeptides in nonequilibrium self-assembly for nanostructure formation.
• To control the recruitment and organization of other proteins, such as collagen, within the assembled structures.

Main Methods:

• Utilized enzyme-responsive phosphopeptides that self-assemble into nanotubes.
• Employed enzyme action to induce shape changes and drive vertical growth of protein nanopillars.
• Leveraged peptide nanotubes as templates to remodel fibronectin and recruit collagen.
• Analyzed structures using Cryo-electron microscopy (Cryo-EM).

Main Results:

• Successfully created cell-free vertical fibronectin pillar mimics via nonequilibrium self-assembly.
• Demonstrated enzyme-catalyzed growth of protein nanopillars into bundles, templated by nanotubes.
• Showcased recruitment of collagen, forming aggregates or bundles based on collagen type.
• Observed nanotube thinning and packing post-dephosphorylation via Cryo-EM, indicating complex assembly dynamics.

Conclusions:

• Established a novel cell-free approach for constructing directed, multiprotein nanostructures.
• Highlighted the role of enzyme-catalyzed nonequilibrium self-assembly in building complex nanoscale architectures.
• Provided insights into the dynamic sculpting processes during peptide and protein assembly.