(723b) “Dock and Lock” Covalent Assembly of Designer Cellulosomes
In this study, we developed a âdock and lockâ method to enable irreversible assembly of cellulosomes by introducing a covalent bond to the cohesin-dockerin interaction. Specifically, homology modeling, docking, and structural analysis were performed to design cohesins and dockerins with single cysteine mutations so that cohesin-dockerin guided assembly causes the formation of a disulfide bond between the two proteins. Our data showed that introducing the cysteine mutations did not affect the cohesin-dockerin interaction, but created a covalent complex that has a greatly reduced dissociation rate and much improved stability without dissociation even under boiling conditions. As a result, these engineered cohesin-dockerin pairs improved the assembly efficiency of designer cellulosomes, which is further translated to enhanced catalytic properties. The method reported here provides a simple way to form covalent designer cellulosomes and can be seamlessly incorporated into any large protein assemblies with minimal design implications.