Eaton, Anna KolesarThe investigation of signal transduction pathways is critical to the basic understanding of cellular processes as these pathways function to regulate diverse processes in both eukaryotes and prokaryotes. This dissertation focuses on understanding some of the biochemical events that take place in the chemotaxis signal transduction pathway of bacteria. In this system, cell-surface receptor proteins regulate a histidine protein kinase, CheA, that autophosphorylates and then transfers its phosphate to an effector protein, CheY. Phospho-CheY, in turn, influences the direction of flagellar rotation. This sequence of biochemical events establishes a chain of communication that ultimately allows the chemotaxis receptor proteins to regulate the swimming pattern of the bacterial cell when it encounters gradients of attractant and repellent chemicals in its environment. The three projects presented in this dissertation sought to fill basic gaps in our current understanding of CheA and CheY function. In the first project, I examined the nucleotide binding reaction of CheA using the fluorescent nucleotide analogue, TNP-ATP [2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate]. TNP-ATP is an effective inhibitor for CheA. By monitoring the fluorescence of TNP-ATP when it bound to CheA, I examined the affinity of the binding interaction and discovered that the two ATP binding sites of each CheA dimer exhibited negative cooperativity in their interactions with TNP-ATP. This is the first evidence of cooperativity in the histidine protein kinase superfamily. In the second project, I focused on elucidating the binding mechanism that underlies formation of the CheA:TNP-ATP complex. My results indicated a three-step mechanism, including rapid formation of a low-affinity complex, followed by two steps during which conformational changes give rise to the final high-affinity complex. This same basic mechanism applied to CheA from Escherichia coli and from Thermotoga maritima. In the third project, I turned my attention to studying the CheY phosphorylation and binding reactions using fluorescently labeled versions of CheY. The results of this final study indicated that CheY proteins labeled with the fluorophore Badan [6-bromoacetyl-2-(dimethylamino)naphthalene] could be useful tools for investigating CheY biochemistry. However my results also brought to light some of the limitations and difficulties of this approach.en-USDissertationBiology, MolecularBiology, MicrobiologyChemistry, BiochemistrychemotaxiskineticsTNP-ATPcooperativityMATLABCheA