Protein cages are a common architectural motif used by living organisms to compartmentalise and control enzymatic processes. While engineered protein cages have recently been featured in the construction of nanoreactors and synthetic organelles, relatively little is known about the fundamental structural features that govern cage stability and molecular flux through their pores.
In this talk, I will outline our latest investigations into the encapsulin family of prokaryotic protein cages.1,2 We have systematically designed a 24-member library of sequence variants based on the T. maritima encapsulin, each featuring pores of different size and charge, as observed in seven new cryo-EM structures of pore variants elucidated at resolutions between 2.5-3.6 Å. Complementing these protein structures, we have used a combination of dynamic light scattering, molecular dynamics, and stopped flow kinetics, to uncover new insights into how structure and flexibility determines the kinetics of such encapsulated nanoreactor systems, paving the way towards programmable synthetic organelles for controlling enzymatic pathways inside living cells.