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Proteins are the workhorses of the cell and support all cellular functions governing biological life. The diversity of protein function is intimately linked to the unique threedimensional structure and intrinsic dynamics of each individual protein. One class of proteins, kinases, are enzymes that catalyze the transfer of a phosphoryl group from ATP to substrate molecules, thereby playing pivotal roles in cellular processes. There are different types of kinases acting on substrates ranging from a simple nucleotide to an entire protein. Aurora B  is a protein kinase with essential roles in cell division and is regulated by multiple mechanisms that ensure faithful mitotic progression. Adenylate kinase (AK) is a small nucleotide kinase that maintains the energy homeostasis in the cell by interconverting ATP and AMP into two ADP molecules.  In this thesis, the link between function, dynamics and structure has been explored in the two kinases Aurora B and AK by combining a selection of structural and functional studies. 

Studies on Aurora kinase B were focused on overcoming production limitations and exploring the effects of phosphorylation on protein dynamics and function. In Paper I, we developed a new production approach of human Aurora B in complex with its regulatory partner inner centromere protein (INCENP) in order to address previous production limitations. The developed approach was based on a fusion protein design with robust expression levels in E. coli, resulting in pure and fully functional protein with yields on the mg-scale. This approach enabled further structural and functional studies and was used to study the effects of phosphorylation on dynamics and substrate binding in Paper II. In this latter paper, we conducted a comparative study between the phosphorylated and dephosphorylated Aurora B:INCENP complex by probing protein dynamics with ¹⁹F NMR spectroscopy experiments. We found that the complex exists in a conformational equilibrium between inactive and active structural states, which is greatly influenced by phosphorylation and substrate binding.

The studies of AK aimed at understanding how small structural elements, such as termini of α-helices and protein bound water molecules, possibly facilitate catalysis. In Paper III, we investigated whether the termini of α-helices could support the large-scale conformational changes underlying catalysis in AK. We employed a comparative approach focused on a bacterial, archaeal and human AK, and found through a combination of X-ray crystallography, NMR spectroscopy and enzymatic activity assay experiments, that the inherent flexibility in the termini of α-helices influences the conformational changes correlated to catalysis. In Paper IV, the role of water molecules in AK catalysis was investigated by swapping the solvent from water to deuterium oxide and measure the effects on catalysis, substrate binding and protein stability with NMR spectroscopy, ITC and CD spectroscopy experiments. We showed that water molecules have an impact on AK catalysis, structural stability and coordination of indirect substrate contacts, all combined suggesting an indirect but important role of water molecules in the phosphoryl transfer reaction. Overall, this thesis contributes to an advanced understanding of how protein structure and dynamics modulate the catalytic function of kinases.