Carboxysome Shell Protein CcmK2 Assembles into Monodisperse and pH-Reversible Microparticles.

Synthetic nano- and microparticles have become essential tools in biotechnology. Protein-based compartments offer distinct advantages over synthetic particles, such as biodegradability and biocompatibility, but their development is still in its infancy. Bacterial microcompartments (BMCs) are protein-based organelles consisting of a protein shell encapsulating an enzymatic core. BMCs are self-assembling, selectively permeable, and modular, making them ideal candidates for the development of protein compartments for biotechnology. Indeed, several groups have engineered BMC shells and individual shell proteins into synthetic nanoreactors and functionalized molecular scaffolds. Expanding the variety of architectures assembled from BMC shell proteins will increase their versatility as building blocks in biotechnology. Here, we developed a method for the in vitro assembly of single-component monodisperse microparticles using only CcmK2, the major hexameric shell protein of the β-carboxysome BMC. We report the controlled assembly of a single type of BMC shell protein into a solid microparticle. High-resolution imaging revealed CcmK2 particles to be assemblies of radially clustered nanotubes. Through biochemical characterization, we determined the conditions for reversible assembly and residues mediating assembly. We found that pH is a key regulator of final particle size and disassembly. Our study situates CcmK2 particles as precisely controlled and self-assembling monodisperse solid protein particles for future applications in biotechnology.