Enhancing the Stability of Bacterial Microcompartments with Natural and Engineered Covalent Crosslinking

Bacterial microcompartments (BMCs) are large protein complexes that are important for metabolic processes in prokaryotes. Structurally, BMCs consist of a polyhedral proteinaceous shell surrounding specific interior enzymes. In cyanobacteria, BMCs called carboxysomes are involved in CO2 fixation, and in enteric bacteria, BMCs called metabolosomes are responsible for propanediol and ethanolamine metabolism. Unfortunately, details of BMC function have remained elusive because purification and subsequent biochemical studies of BMCs have been a challenge due to their low stability in solution. We aim to explore specific covalent crosslinking between shell protein components as a method for improving the stability of BMCs to allow purification and in vitro studies. In carboxysomes from the cyanobacterium Synecococcus elongatus, we used site-directed mutagenesis to engineer specific cysteine residues as sites for modification with covalent crosslinkers. We found that carboxysome shell proteins containing engineered cysteine mutations can be modified with covalent crosslinkers in vitro. Additionally, using reducing and non-reducing PAGE gels, we discovered intermolecular disulfide bonds between native cysteine residues in a BMC shell protein from Clostridia, which connect neighboring shell protein subunits. As judged by electrophoretic mobility shift, this shell protein shows complex patterns of disulfide bond formation between monomeric subunits. Overall, our work demonstrates that both engineered and naturally-occurring disulfide bonds can be used to stabilize BMCs in solution. Utilization of BMCs with enhanced stability will allow purification and in vitro manipulation of these elaborate protein complexes, thus enabling new experiments that will expand our understanding of BMC function.