Bacterial cells possess significantly more ultrastructural organization than is typically appreciated. Oneof the most striking examples of this are bacterial microcompartments (BMCs), large (i.e. 100+ nm)proteinaceous complexes that encapsulate cargo enzymes catalyzing a short metabolic pathway withina capsid-like shell. BMCs enable metabolism incompatible with their host and this functional advantageis borne out in their pervasiveness. 20-30% of bacterial genomes possess BMC-like proteins. Despitethis prevalence, only a handful of BMCs are characterized. One of the most intriguing open questionssurrounding BMCs is how a mature functional complex emerges from only protein-protein interactions.Specifically, the mechanism of assembly, cargo ordering and stoichiometry, and the robustness, shape,and size of the mature complex cannot be explained from the current qualitative knowledge of knownprotein interactions. The goal of our work is to use mechanistic biochemical approaches in order tounderstand the in vivo self-assembly and function of the BMC known as the ?-carboxysome (?-CB). The?-CB facilitates autotrophic growth in many bacteria and was the first BMC to be characterized due to itsrobustness and ease of biochemical analysis. It is therefore an excellent model system to answer theseopen questions. Preliminary data indicates that a protein known as CsoS2 is essential for ?-CB formationand may be the hub of an interaction network driving self-assembly. We propose to use biochemical andbiophysical tools in order to both map the molecular determinants of these interactions and quantitativelyunderstand how multivalency controls assembly. CsoS2 is also an intrinsically disordered protein andpossesses numerous repetitive sequence elements. Preliminary data indicates these regions of CsoS2play an important role in determining ?-CB size. Intrinsically disordered proteins are known to participatein an organizing role in eukaryotes, but are largely uncharacterized in prokaryotes. We therefore proposea series of experiments to understand the significance of disorder to CsoS2 function and how its repetitiveelements are involved in determining the outcome of the assembly process. Finally, it has long beenpostulated that BMCs act like an organelle and possess a chemical environment that is distinct from thecytosol. This hypothesis is supported by circumstantial data but has never been directly measuredbiochemically due to experimental challenges. Here we proposed a series of experiments to make thismeasurement ex vivo by determining whether the ?-CB naturally possesses an oxidative lumen due tothe action of its protein shell. We will additionally determine to what extent the chemistry of the lumenaffects the self-assembly process. If successful, these experiments will provide novel mechanistic insightinto how BMCs assemble and function, and more broadly, the interplay between bacterial ultrastructureand bacterial physiology.