Chemistry & Biochemistry Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2752
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Item An investigation of allosteric mechanisms in biotin protein ligases using integrated biophysical approaches(2019) Wang, Jingheng; Beckett, Dorothy; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Allostery is a biological process in which action, often ligand binding, at one site of the protein alters the function at another site. It provides a mechanism for modulating protein functions in a variety of cellular events ranging from signaling, metabolism, to transcription regulation. Despite the critical role of allostery in biology and intense research during the past few decades, the mechanism of long-range communication through the protein is still elusive. The Escherichia coli biotin protein ligase (BirA) is a bifunctional protein that catalyzes post-translational biotinylation and represses transcription initiation. It serves as a model system to investigate long-range allosteric communication, as binding of the effector molecule, bio-5’-AMP, promotes the repressor complex assembly by enhancing BirA homodimerization occurring at a surface 30Å away. Previous studies have established that disorder-to-order transitions of several loop segments on the ligand binding and dimerization surfaces contribute to BirA allostery. In this dissertation, integrated structural, functional, and computational approaches were used to investigate the molecular mechanisms of allosteric communication between these transitions. Double-mutant cycle analysis demonstrated reciprocal coupling between residues on two distant surfaces, and results of molecular dynamics simulations indicated that functional coupling occurs via modulation of structure and dynamics of surface loops undergo disorder-to-order transitions. Further structural and simulation-based network analyses revealed that these transitions are linked to formation of a residue network, and alanine substitutions of residues at network positions perturb both input (effector binding) and output (dimerization) of allostery. In addition, Force Distribution Analysis showed that perturbed loop folding is associated with redistribution of mechanical stress experienced by network residues. The combined results indicated a mechanism for BirA allosteric regulation in which disorder-to-order transitions and joint network formation enables long-range communication through the protein. Finally, results of functional measurements indicated a conserved allosteric regulation mechanism among Escherichia coli (Ec), Staphylococcus aureus (Sa), and Bacillus subtilis (Bs), as bio-5’-AMP binding to Sa and BsBirA induces homodimerization similar to that observed for EcBirA.Item A Thermodynamic Investigation into the Allosteric Activation Mechanism of the Biotin Repressor(2004-11-05) Brown, Patrick H.; Beckett, Dorothy; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The 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.