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A Thermodynamic Investigation into the Allosteric Activation Mechanism of the Biotin Repressor

dc.contributor.advisorBeckett, Dorothyen_US
dc.contributor.authorBrown, Patrick H.en_US
dc.description.abstractThe biotin regulatory system of Escherichia coli serves as a model for investigating the regulatory mechanism of a non-classical allosteric transcription factor. The central protein, BirA, functions as both an essential metabolic enzyme in biotin retention and as a repressor of transcription initiation. In its repressor function, two BirA monomers bind a 40-base pair palindromic DNA sequence thereby blocking transcription initiation at the two divergent overlapping promoters of the biotin biosynthetic operon. Binding of the small molecule corepressor, biotinyl-5'-AMP, promotes the assembly of the transcription repression complex by driving the self-association of the repressor. Here, the effects of binding of four corepressors on the self-association and DNA binding properties of BirA have been measured utilizing sedimentation equilibrium and DNaseI footprinting analyses. The results of this study indicate that biotinyl-5'-AMP and an ester analog, biotinol-AMP, are strong allosteric activators of BirA dimerization. The enhancement observed in the energetics of DNA binding closely matches with the enhancement of self-assembly of the repressor. Biotin and a sulfamoyl corepressor analog are weak allosteric effectors of BirA dimerization. Binding of the weak effectors, results in an uncoupling of the self-association and DNA binding processes. A detailed thermodynamic investigation of the effector binding process was performed utilizing isothermal titration calorimetry. Binding of all four corepressors to BirA is an enthalpically driven process. However, the higher affinities for binding of the strong effectors are characterized by a relatively moderate binding enthalpy and a favorable entropic term. Whereas binding of the weak effectors is comprised of a much larger enthalpic contribution and is entropically opposed. Heat capacity changes for binding of the four effectors to BirA were determined by measuring the temperature dependence of the binding enthalpy. Results of the analysis indicate that a negative heat capacity change is associated with binding of each effector. No correlation is observed between the magnitude of the heat capacity change and the magnitude of the effect the corepressor has on the self-assembly of BirA. Finally, conditions were identified and utilized for the crystaliztion of BirAbiotinol-AMP. The crystals obtained are currently being analyzed by X-ray diffraction in a collaborative effort.en_US
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dc.titleA Thermodynamic Investigation into the Allosteric Activation Mechanism of the Biotin Repressoren_US
dc.contributor.publisherDigital Repository at the University of Marylanden_US
dc.contributor.publisherUniversity of Maryland (College Park, Md.)en_US
dc.subject.pqcontrolledChemistry, Biochemistryen_US
dc.subject.pquncontrolledthermodynamics of activationen_US
dc.subject.pquncontrolledBiotin Repressoren_US
dc.subject.pquncontrolledtranscription repressoren_US

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