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dc.contributor.authorRuangprasert, Ajchareeya
dc.date.accessioned2014-03-04T18:57:35Z
dc.date.available2014-03-04T18:57:35Z
dc.date.issued2010-08
dc.identifier.otherruangprasert_ajchareeya_201008_phd
dc.identifier.urihttp://purl.galileo.usg.edu/uga_etd/ruangprasert_ajchareeya_201008_phd
dc.identifier.urihttp://hdl.handle.net/10724/26773
dc.description.abstractBacterial transcriptional regulators have been identified as potential targets for the development of novel antibiotics. LysR-type transcriptional regulators (LTTRs), which comprise the largest group of transcriptional regulators in proteobacteria, regulate diverse metabolic functions including antibiotic resistance and virulence factor synthesis rendering them potential drug targets. Molecular characterization of LTTRs is crucial for the understanding of LTTRs’ mechanism of regulation. LTTRs consist of a DNA-binding domain (DBD), a linker helix (LH), and an effector binding domain (EBD) that is composed of two subdomains (EBD-I and EBD-II). BenM, an LTTR found in Acinetobacter baylyi ADP1 provides a well-characterized model for structural studies of LTTRs. The structures of full-length BenM and two constitutively active mutants, BenM(E226K) and BenM(R156H), determined by X-ray crystallography illustrate an unexpected “infinite” oligomer of the regulators in their crystal lattices. In addition, all three of the structures are nearly identical suggesting an undefined mechanism that triggers the different in vivo activity of these proteins rather than simple conformational changes as a result of amino acid differences. Analysis of BenM with other LTTRs revealed that there are at least three major oligomerization schemes used by LTTRs. One mode represented by CbnR, DntR and BenM utilizes EBD-II/EBD-II and DBD contacts, a second mode represented by 2ESN, ArgP and TsaR utilizes only DBD contacts for tetramerization, and the third mode utilizes both DBD and EBD-I/EBD-II contacts and is represented by 3FZV. Effector-binding and DNA-binding studies of BenM and its variants confirmed the functionality of the purified proteins used in the crystallization and provided additional insight into the biophysical properties of BenM. The dissociation constants (Kd) of BenM and BenM variants for the effectors, benzoate and cis,cis-muconate were derived by tryptophan fluorescence spectroscopy. Affinities of BenM, BenM(E226K) and BenM(R156H) for their target promoters were investigated by electrophoretic mobility shift assay (EMSA).
dc.languageeng
dc.publisheruga
dc.rightspublic
dc.subjectLysR-type transcriptional regulator
dc.subjectBenM
dc.subjectAcinetobacter
dc.titleStructural characterization and biophysical studies of BenM, a LysR-type transcriptional regulator in Acinetobacter baylyi ADP1
dc.typeDissertation
dc.description.degreePhD
dc.description.departmentPharmaceutical and Biomedical Sciences
dc.description.majorPharmacy
dc.description.advisorCory Momany
dc.description.committeeCory Momany
dc.description.committeeRobert Phillips
dc.description.committeeEllen L. Neidle
dc.description.committeeShelley Hooks
dc.description.committeeAnthony Capomacchia


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