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dc.contributor.advisorGiedroc, David P.
dc.contributor.advisorSacchettini, James C.
dc.creatorReyes Caballero, Hermes
dc.date.accessioned2012-10-19T15:28:32Z
dc.date.accessioned2012-10-22T17:59:01Z
dc.date.available2012-10-19T15:28:32Z
dc.date.available2012-10-22T17:59:01Z
dc.date.created2011-08
dc.date.issued2012-10-19
dc.date.submittedAugust 2011
dc.identifier.urihttp://hdl.handle.net/1969.1/ETD-TAMU-2011-08-9738
dc.description.abstractNmtR and AdcR belong to two structural and functional classes of transcriptional metalloregulators. The present study shows that AdcR is a novel Zn(II) dependent repressor and the first ever metalloregulator of the MarR family. In contrast, NmtR is a repressor that is inactivated by Ni(II) binding. NmtR is a member of the extensively characterized ArsR/SmtB family. Two Ni(II) ions bind to the NmtR dimer in an octahedral coordination complex with stepwise binding affinities of KNi1 = 1.2 (±0.1) x 10¹⁰ and KNi2 = 0.7 (±0.4) x 10¹⁰ M⁻¹ (pH 7.0). A glutamine scanning mutagenesis approach reveals that Asp 91, His 93, His 104 and His 107 in the [alpha]5 helix and His 3 at the extreme N-terminal contribute to KNi. In contrast residues from the C-terminal tail (H109, D114 and H116), previously implicated in NmtR binding, are characterized by near wild-type KMe and allosteric coupling free energies. However, deletion of most of the C-terminal tail to create Δ111 NmtR reduce Ni(II) binding stoichiometry to one per dimer and greatly reduced Ni(II) responsiveness. H3Q and Δ111 NmtR also show important perturbations in the rank order of metal responsiveness, with both different from wild-type NmtR. The use of both presumably unstructured N- and C- terminal extensions is a unique property relative to other members of the ArsR/SmtB family previously characterized and provides a distinct metal specificities profile. AdcR binds two regulatory Zn(II) ions per dimer in an unusual five coordinate geometry as determined by X-ray and electronic absorption spectroscopy. Functional characterization of single residue substitution mutants identified His 108 and His 112 in [alpha]5 helix and His 42 in [alpha]2 helix, as residues essential for allosteric activation of DNA operator binding by AdcR as revealed by fluorescence anisotropy experiments. The stability constant for the regulatory site, KZn, is sensitive to pH and range from ~10¹⁰ M⁻¹ at pH 6.0 to ~10¹⁰ M⁻¹ at pH 8.0. Zn(II) binds to an additional one to two pairs of ancillary sites per dimer depending on the pH. A novel feature not shared by other Zn(II) regulators is an apparent reduced metal specificity, since non-cognate metals Mn(II) and Co(II) activate AdcR to the same extent than Zn(II) does. However, each non-cognate metal binds with very low affinity (<̲ 10⁶ M⁻¹ at pH 8.0) and are not inducers in vivo.en
dc.format.mimetypeapplication/pdf
dc.language.isoen_US
dc.subjectNmtRen
dc.subjectAdcRen
dc.titleBiochemical Characterization of the Mycobacterium tuberculosis Ni(II) Sensor NmtR and Streptococcus pneumoniae Zn(II) Sensor AdcRen
dc.typeThesisen
thesis.degree.departmentBiochemistry and Biophysicsen
thesis.degree.disciplineBiochemistryen
thesis.degree.grantorTexas A&M Universityen
thesis.degree.nameDoctor of Philosophyen
thesis.degree.levelDoctoralen
dc.contributor.committeeMemberLindahl, Paul A.
dc.contributor.committeeMemberKapler, Geoffrey M.
dc.type.genrethesisen
dc.type.materialtexten


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