Elucidation of genes of unknown function in alpha carboxysome operons: acRAF, BMVs and carbon regulatory PII proteins.
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.