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dc.contributor.authorGreenswag, Annaen_US
dc.date.accessioned2015-04-06T20:13:33Z
dc.date.available2020-01-27T07:00:30Z
dc.date.issued2015-01-26en_US
dc.identifier.otherbibid: 9154378
dc.identifier.urihttps://hdl.handle.net/1813/39301
dc.description.abstractBacterial chemotaxis enables changes in motility via response to the surrounding chemical environment and is noted for its high signal gain, range, and sensitivity. The efficacy of the bacterial chemotaxis signaling pathway is highly dependent on the propagation of the extracellular chemical signal through a hexagonal array comprised of: methyl-accepting chemotaxis protein receptors, histidine kinase CheA, and coupling protein CheW. CheA is the principal enzyme in the chemotaxis pathway and is composed of five domains (P1-5). Initiation of the phospho-relay by CheA ends in rotational switching of the flagellar rotor. CheA only achieves a broad range of autophosphorylation activity when associated with chemoreceptors. This dissertation focuses on the structural and biochemical changes during the CheA autophosphorylation event. The propensity of Thermotoga maritima CheA to naturally undergo trans autophosphorylation was elucidated and strategic mutations enabled generation of disulfide-locked CheA variants to further probe protein dynamics. Employing small-angle x-ray scattering (SAXS), the resting state of CheA was determined to be globular. Where upon nucleotide addition the protein transitioned to a dynamic state as a result of the movement of P1 and P4 domains to facilitate transfer of the [gamma]-phosphate. Coupling crystallographic and biochemical data, a model was generated of CheA that is able to account for variances in enzymatic activity, incorporating key structural features to the functional response of signal transduction. To further the understanding the influence the receptors impart to CheA, chemoreceptor cytoplasmic kinase-control modules based on the E. coli aspartate receptor, Tar, were covalently fused into a dimer and trimerized by a foldon domain (TarFO). SAXS, multiangle light scattering, and pulsed-dipolar electron paramagnetic resonance spectroscopy of spinlabeled proteins indicate that the TarFO is soluble, monodisperse, and assembles into homogenous trimers wherein the protein interaction regions closely associate at the opposite ends of the foldon domain. The TarFO activates CheA autophosphorylation to the same degree as membrane integrated receptors and stabilizes a planar conformation of the kinase consistent with current array models for the assembly state of the ternary complex. Overall, these studies illuminate a planar CheA active structure and provide a more in depth investigation of the CheA autophosphorylation event.en_US
dc.language.isoen_USen_US
dc.titleStructure And Enzymology Of The Chemotaxis Histidine Kinase, Cheaen_US
dc.typedissertation or thesisen_US
thesis.degree.disciplineChemistry and Chemical Biology
thesis.degree.grantorCornell Universityen_US
thesis.degree.levelDoctor of Philosophy
thesis.degree.namePh. D., Chemistry and Chemical Biology
dc.contributor.chairCrane, Brianen_US
dc.contributor.committeeMemberLin, Heningen_US
dc.contributor.committeeMemberCerione, Richard Aen_US


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