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All life on Earth relies on biological carbon fixation, the process by which organisms convert inorganic carbon, primarily in the form of carbon dioxide (CO2), into longer chain compounds to fuel cellular processes. To enhance the efficiency of CO2 fixation, certain types of bacteria, specifically cyanobacteria and some proteobacteria, evolved specialized proteinaceous microcompartments called carboxysomes. Carboxysomes encapsulate the enzymes carbonic anhydrase and Rubisco inside a polyhedral layer of shell proteins. This molecular architecture serves to concentrate CO2 around Rubisco, allowing it to operate at its maximum catalytic rate.Correct carboxysome assembly is essential to the survival of the organism in CO2 concentrations found in today’s atmosphere (~0.04%). In the ⍺-carboxysome lineage, the disordered scaffold protein CsoS2 links Rubisco and shell proteins, and is absolutely required for carboxysome formation and cell growth at ambient CO2 levels. This work examines how the sequence of CsoS2 scales from a disordered amino acid chain to directing the ordered self-assembly of thousands of proteins. It investigates how cells utilize specific chemistries, such as redox reactions, to assist in this assembly pathway. The result of this molecular design and coordinated construction is to build a carboxysome with a precise permeability, yet this permeability has never been measured. Results presented here address these fundamental questions.I interrogated highly conserved and repetitive residues in CsoS2 to determine their role in carboxysome assembly. Through in vivo mutagenesis and in vitro biochemical assays I discovered that the residues VTG and Y are necessary for carboxysome assembly, and bind weakly yet multivalently to shell proteins. Conserved cysteine doublets, which hinted at a role for redox in assembly, showed no effect when mutated in vivo, but displayed biochemical phenotypes in vitro. In a major step towards reconstituting carboxysomes in vitro, I demonstrated formation of carboxysome-like phase-separated condensates with Rubisco, CsoS2, and shell, thereby showing that key carboxysome proteins can self-associate in a cell-free environment.Once assembled correctly, the carboxysome establishes a permeability barrier and selectivity filter, allowing entry of essential metabolites such as ribulose bisphosphate and bicarbonate while restricting leakage of CO2. To measure carboxysome permeability, we developed two parallel methods, one based on a bulk plate assay and one on single-particle microscopy. Both methods utilized the redox sensitive reporter protein roGFP to simultaneously measure both the permeability of reducing agents and the internal carboxysome redox environment. Data from both approaches revealed that purified carboxysomes were permeable to the reducing agent TCEP, which reduced encapsulated roGFP over time.Carboxysomes are the bacterial domain’s solution to the problem of capturing dilute CO2 from air and water, concentrating it, and converting it into sugars. Carboxysome functionality depends on the robust self-assembly of thousands of proteins, establishment of a specific internal chemical environment, and control over metabolite permeability. Insights from this work augment our understanding of these processes, and will aid future efforts to engineer carboxysomes into alternative organisms or cell-free systems for enhanced biological carbon capture.

Photosynthesis encompasses the set of reactions that convert light into stored chemical energy. Photosynthetic organisms are the foundation of life, as they catalyze the assimilation of inorganic carbon (Ci) into the biosphere and are the ultimate source of nutrition on Earth. Cyanobacteria are an ancient group of oxygenic photosynthetic prokaryotes and remain important primary producers today. The chloroplasts of all land plants can trace their ancestry to cyanobacteria. In many ways, such as the light reactions of photosynthesis that capture solar energy, the protein machinery of cyanobacteria is a representative, simplified form of that found in chloroplasts. In other ways, the cyanobacterial machinery is more complex as in the dark reactions that ultimately store solar energy as chemical energy by fixing CO2. To overcome inherent limitations in the principal CO2-fixing enzyme rubisco, cyanobacteria possess an elaborate integrated physiological strategy to increase the local CO2 concentration near rubisco and facilitate on-pathway catalysis. This physiological strategy, known as a CO2-concentrating mechanism (CCM), relies on energy-coupled Ci-transporters and encapsulation of rubisco in proteinaceous organelles known as carboxysomes. While some species of land plants possess CCMs, none resemble nor are nearly as productive as those found in cyanobacteria. Therefore, cyanobacteria are an ideal platform to study the basic workings of photosynthetic machinery and potentially provide insight on engineering strategies that may improve photosynthetic productivity in land plants of societal interest. Here, we investigated the structure and function of protein complexes from cyanobacteria and other prokaryotes to elucidate their roles in photosynthesis and CCMs. First, we determined the structure of the NAD(P)H dehydrogenase-like complex, a key component in the light reactions, and develop putative models for its catalytic mechanism. Next, we discovered a new type of Ci-transporter we named the DABs accumulate bicarbonate (DAB) complex and characterize the complex through a variety of genetic, biochemical, and biophysical approaches. Finally, we determined the structure of simplified carboxysome shells that provide insight into the architecturally principles and assembly of native shell components.

The chapters of this thesis describe the structure and function of three proteins involved in assembly and function of bacterial microcompartments. Bacterial microcompartments (MCPs) are often thought of as bacterial organelles; they are proteinaceous structures that spatially compartmentalize metabolic reactions for increased catalytic efficiency. There are three types of characterized MCPs: those that catalyze either the catabolic utilization of propanediol (Pdu MCP), ethanolamine (Eut MCP), or carbon dioxide (called carboxysomes). Chapter 1 describes studies of bacterial microcompartment vertex proteins (BMVs) from various microcompartments. BMVs are thought to occupy the 12 vertices of the MCP icosahedron capsule. However, past structural studies of the BMV from E. coli, EutN, reveal pseudo–hexagonal shapes, whose geometry is unfit for incorporation into pentagonal vertex spaces of microcompartments. We developed a novel method called OCAC (Oligomeric Characterization by Addition of Charge) to probe BMV oligomeric states in solution. We also endeavored to determine the oligomeric states of the two conserved BMV proteins from alpha–carboxysome operons. The reason for the existence of paralogous BMV proteins in alpha–carboxysome is not understood. The contents and figures of Part–I of Chapter–I were published in Protein Science in 2013 (1). Yuxi Lui performed the experiment reported in Figure 2. Soheil Gidaniyan and Nicole Wheatley performed protein expression, purification and crystallization of GrpN and other required proteins. Duilio Cascio provided assistance in solving the GrpN crystal structure. Nicole Wheatley and Todd Yeates coordinated and designed experiments and prepared the manuscript. The contents of Part–II have not been published. Joanna Ngo expressed, purified and performed gels shown in that chapter sub–section. Nicole Wheatley designed and coordinated efforts. Chapter 2 describes the discovery of a novel Rubisco (Ribulose–1,5–bisphosphate carboxylase/oxygenase) chaperone encoded within the operons of alpha–carboxysomes. We named this distant pterin–4a–carbinolamine dehydratase homolog acRAF, for alpha carboxysome Rubisco Assembly Factor. Heterologous co–overexpression of acRAF, GroELS and CbbLS yields greatly increased amounts of soluble, folded Rubisco compared to co–overexpression with GroELS and CbbLS alone. The contents and figures of Chapter–II were published in the Journal of Biological Chemistry in 2014 (2). Christopher Sundberg created the DNA constructs used in Figure 5 of that publication, or Figure 9 of this thesis. Soheil Gidanyian and Nicole Wheatley performed protein expression, purification and crystallization experiments on acRAF. Nicole Wheatley performed the experiment shown in Figure 10A and AB of this thesis. Soheil Gidanyian performed experiment shown in Figure 10C. Duilio Cascio assisted in solving the crystal structure of acRAF. Todd Yeates made Figure 7A. Nicole Wheatley and Todd Yeates prepared the manuscript. Nicole Wheatley designed and coordinated the efforts of the work. Chapter 3 describes a nitrogen regulatory PII–like protein encoded within several proteobacterial alpha–carboxysome. Crystal structures of this protein, here named ndhPII, display several unique characteristics compared to canonical nitrogen regulatory PII proteins. We structurally analyze both unbound and ADP–bound ndhPII crystal structures, and speculate about its regulation of inorganic carbon assimilation in these organisms. The work contained in this chapter is not yet published. Joanna Ngo and Nicole Wheatley performed protein expression, purification and crystallization of ndhPII. Duilio Cascio and Michael Sawaya solved and refined both crystal structures. Nicole Wheatley designed and coordinated the efforts of this work.