Composed entirely of protein subunits, protein cages are porous, self-assembling structures that are found in a broad range of microorganisms. In nature, protein cages in the form of viral capsids and bacterial microcompartments perform a diverse array of functions – from encapsulating genomic material to coordinating metabolic pathways within the cell.1,2 More recently, protein cages have been engineered to serve as templates for synthetic organelles, reaction chambers and sensing devices.3–6 Central to these applications are the pores that perforate the protein cage. However, a systematic study investigating the effect of pore composition on overall cage assembly, stability and molecular diffusion remains yet to be done.
Here we re-designed the pore architecture of a class of protein cages known as encapsulins. We used the encapsulin protein cage from the bacterium, Thermotoga maritima7 and designed a 24-member variant library by altering the pore size and/or charge. We then systematically examined the effects of both the structure and chemical nature of the pore on cage assembly, stability and molecular flux of cations into the cage. We report on twelve designs that were successfully purified, of which eight were found to exhibit prolonged thermal stability. Together with seven cryo-EM structures of cage variants and kinetic studies for quantifying cation influx into the cage, we analyse how the pore architecture governs cage stability and molecular recognition at the pore.