| In the bacterial chemotaxis pathway, a receptor kinase complex is formed by three proteins known as chemoreceptor, CheA and CheW. The autokinase CheA is coupled to chemoreceptors via the coupling protein CheW. This thesis focuses on the intermolecular interaction between CheA and CheW as well as intramolecular interactions within CheA.; Using the CheA/CheW interaction as a model system, I have developed a new method to map the residues at a protein-protein interface for macromolecular complexes of molecular weight >100 kDa. The essence of the idea is that a portion of the intensity of HMQC spectra of individual -13CH 3 resonances in an otherwise deuterated macromolecule have much reduced dipole-dipole relaxation and remain sharp and relatively easy to detect, even in macromolecules of molecular mass 100 kD or greater. The reduction in dipolar interactions is lost if a given methyl group comes in close contact with other protons such as those supplied by the interface of a protonated interaction partner. Conversely, if the interaction partner is fully deuterated, the resonance of the methyl group in question remains relatively sharp. Thus, by comparing the 13CH3 resonances of a protein of interest in the presence of a protonated versus deuterated interaction partner, the methyls at the interface can be identified. This methodology has been successfully implemented to define the residues in CheW located at the binding surface for CheA.; The CheA autophosphorylation reaction minimally involves two CheA domains, denoted P1 and P4. The kinase domain (P4) binds ATP and orients the gamma-phosphate for phosphotransfer to a reactive histidine on the phosphoacceptor domain (P1). Here, NMR chemical-shift-perturbation-mapping as well as distance restraints from paramagnetic relaxation describe the interaction of P1 with P4. The two methods agree on the P4 surface that binds P1, but implicate opposite surfaces of P1 in P4 binding. Interestingly, both methods suggest the presence of multiple binding modes which may address the ability of CheA to switch rapidly from a binding mode that allows P1 phosphorylation to a binding mode that allows P1 to transfer this phosphate to CheY or CheB. |
