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The BirA functional switch is investigated using DNaseI footprinting and MALDI-TOF mass spectrometry. Results of these measurements indicate that BirA can be selectively targeted toward its enzymatic function by increasing the kinetic probability of heterodimerization relative to that of homodimerization.

Research has been conducted on Escherichia coli biotin repressor. The quentitative relationship between small ligand binding, DNA binding and dimerization in the biotin repressor system has been investigated via DNAseI footprinting, and the results reveal that these species bind to the operator cooperatively and specifically.

The Escherichia coli repressor of biotin biosynthesis, BirA, binds site-specifically to the biotin operator, a 40 base pair imperfect inverted palindrome. Two repressor monomers have been shown to bind to the two operator half-sites. Analysis of results of quantitative DNase I footprint titrations performed on the wild-type biotin operator template indicate that binding is well described by a cooperative mechanism. The data obtained from these studies were, however, insufficient to independently resolve all of the energetic parameters associated with cooperative binding of the two repressor monomers to the operator site. In this work, to further dissect the energetics of assembly of the biotin repressor - biotin operator complex, measurements of binding of BirA to four bioO variants designed to reduce the valency of repressor binding from 2 to 1 have been performed. Results of these measurements indicate, as was found with the wild-type biotin operator template, that two repressor monomers bind simultaneously to the two half-sites of all variant operators. Protein dimerization and DNA binding are thus obligatorily coupled in the biotin repressor system. Furthermore, the results suggest that, in the context of a cooperative binding mechanism, the cooperative free energy associated with the biotin repressor - biotin operator interaction is significantly more favorable than the previously estimated -2 kcal/mol.

The binding of the Escherichia coli biotin holoenzyme synthetase to the two ligands, biotin and bio-5′-AMP, is coupled to disorder-to-order transitions in the protein. In the structure of the biotin complex, a 'glycine-rich' loop that is disordered in the apo-enzyme is folded over the ligand. Mutations in three residues in this loop result in significant changes in the affinity of the enzyme for both biotin and bio-5′-AMP. The kinetic basis of these losses in the affinity resides primarily in changes in the unimolecular rates of dissociation of the complexes. In this work, isothermal titration calorimetry has been employed to examine the detailed thermodynamics of binding of three loop mutants to biotin and bio-5′-AMP. The energetic features of dissociation of the protein•ligand complexes also have been probed by measuring the temperature dependencies of the unimolecular dissociation rates. Analysis of the data using the Eyring formalism yielded entropic and enthalpic contributions to the energetic barrier to dissociation. The thermodynamic results coupled with the known structures of the apo-enzyme and biotin complex have been used to formulate a model for progression from the ground-state complex to the transition state in biotin dissociation. In this model, the transition-state is characterized by both partial disruption of noncovalent bonds and acquisition of some of the disorder that characterizes the glycine-rich loop in the absence of ligand. [ABSTRACT FROM AUTHOR]

The binding of the Escherichia coli biotin holoenzyme synthetase to the two ligands, biotin and bio-5'-AMP, is coupled to disorder-to-order transitions in the protein. In the structure of the biotin complex, a "glycine-rich" loop that is disordered in the apo-enzyme is folded over the ligand. Mutations in three residues in this loop result in significant changes in the affinity of the enzyme for both biotin and bio-5'-AMP. The kinetic basis of these losses in the affinity resides primarily in changes in the unimolecular rates of dissociation of the complexes. In this work, isothermal titration calorimetry has been employed to examine the detailed thermodynamics of binding of three loop mutants to biotin and bio-5'-AMP. The energetic features of dissociation of the protein*ligand complexes also have been probed by measuring the temperature dependencies of the unimolecular dissociation rates. Analysis of the data using the Eyring formalism yielded entropic and enthalpic contributions to the energetic barrier to dissociation. The thermodynamic results coupled with the known structures of the apo-enzyme and biotin complex have been used to formulate a model for progression from the ground-state complex to the transition state in biotin dissociation. In this model, the transition-state is characterized by both partial disruption of noncovalent bonds and acquisition of some of the disorder that characterizes the glycine-rich loop in the absence of ligand.