(65d) Designing Viral Capsid Nanoreactors for the Study of Compartmentalized Processes | AIChE

(65d) Designing Viral Capsid Nanoreactors for the Study of Compartmentalized Processes


Tullman-Ercek, D. - Presenter, University of California, Berkeley

Bacterial microcompartments (BMCs) are bacterial organelles comprising a large icosahedral protein shell that houses metabolic processes, and have great potential to serve as scaffolds for both in vitro and in vivo nanoreactors. For such applications, it is critical that we are able to control the diffusion of small molecules into BMCs. In fact, this may be the primary natural function of the BMC; it is speculated that the protein shell of one BMC, the carboxysome of photosynthetic cyanobacteria, serves to enable an increased local CO2 concentration near the carbon-fixation enzyme, and sequester that enzyme from oxygen. Due to the difficulty of working with carboxysomes, this theory has gone untested. Here, we will discuss our work on developing the well-characterized MS2 viral capsid as a model system to investigate protein membrane permeability. The capsid is spherical with icosahedral symmetry, and has 1.8 nm pores at its 5-fold and quasi-6-fold axes. When expressed recombinantly in E. coli 180 monomers of capsid protein assemble into intact capsid that is stable to a wide range of pH’s and denaturants, in addition to mutations and chemical modifications. We hypothesize that the pores of the capsid (and BMC shell) are the primary control point for small molecule diffusion. To investigate this idea, we are working towards encapsulating enzymes in the capsid while varying the charge around the pore, so that we can use enzymatic activity as a readout of diffusion. This encapsulation requires disassembly of the capsid followed by reassembly around a specific nucleic acid, which is conjugated to our protein of interest. With this presentation, we will describe 1) the effect of various nucleic acids on MS2 reassembly, 2) how we greatly enhanced reassembly yields with the addition of certain small molecules, 3) the bioconjugation methods used to attach the nucleic acids to our model protein, and 4) effects of pore-residue alterations on capsid reassembly.