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Project Description

Bacterial microcompartments (BMCs) are endogenous platforms ideally suited for synthetic biology, as modular protein structures of relatively simple construction. Of the three known BMCs (carboxysomes, PDUs, and EUTs), EUTs were chosen as candidates for photo-mechanization due to their comparatively straightforward assembly. While carboxysomes and PDUs require precise ratios of coexpressed protein subunits to assemble, the ethanolamine utilizing microcompartments’ shell can form in vivo from a single subunit: EutS. Our research focused on the incorporation of azobenzene-sidechain noncanonical amino acids into the EutS protein, which was hypothesized to confer the nanocages with a photo-switchable function for assembly and disassembly.

Photo-mechanized protein nanocages could serve as adaptable tools for a broad spectrum of synthetic biologists and engineers; applications include compartmentally isolated biocatalyses, and targeted cargo transport and delivery (e.g. precise drug delivery). As such, our research has emphasized general optimization of a light-induced nanocage, although avenues to specific applications have been partially paved in the process.

Protein nanocages like EUTs are attractive subjects for engineered modifications, since their shells’ structural uniformity reduces the influence of unknown variables on our understanding of the BMC system. Although bacterially-encoded EUTs contain several shell protein constituent subunits (including EutS, M, N, L, & K), the EutS component is sufficient for complete shell formation in vivo. EutS homohexamerizes, then further complexes with additional homohexamers to form polyhedral nanocages with predictable subunit interfaces and vertices. Rosetta and PyMol were employed in analyzing the thermodynamically favorable interfacing of subunits, and identifying corresponding residues at which azobenzene substitutions wouldn’t disrupt nanocage assembly.

Several candidate residue sites were tested (i.e. AA-encoding codon replaced with amber stop codon; Schultz lab tRNA incorporated azobenzene-moiety amino acids at these sites), and some cages were observed to form uninterrupted with the incorporated azobenzene AAs. While in situ assembly/disassembly has yet to be accomplished, our observations involving pre-irradiated azobenzene indicate that cis-trans isomerization of the noncanonical amino acid does incur subunit conformational changes which overtly affect nanocage assembly.