Development and application of hydrogen/deuterium exchange and mass spectrometry to the thermodynamic analysis of DNA binding proteins and their complexes

The thermodynamic analysis of protein-DNA interactions is important for understanding many important biological processes. Conventional approaches for such analyses have included gel mobility shift assays, filter binding assays and fluorescence anisotropy techniques. These approaches require selective labeling, highly purified samples and they are not generally high-throughput. Recently, a new H/D exchange- and mass spectrometry-based SUPREX technique, (Stability of Unpurified Protein from Rates of H/D Exchange) has been developed. SUPREX is not subject to the above limitations as it affords the unique ability to perform thermodynamic analyses on small amounts of unpurified protein samples in a high throughput fashion. This thesis is focused on extending the application of SUPREX to the thermodynamic analyses of protein-DNA interactions. Several aspects of the technique regarding the experimental protocol and data analysis have also been further developed as part of this work.; As part of this thesis the SUPREX technique was used to study the thermodynamic properties of four DNA binding proteins including TrpR, ArcR, CopG and MetJ. The binding affinities of these proteins to their cognate oligonucleotide sequences and non-cognate oligonucleotide sequences were evaluated by measuring the changes in folding free energies (i.e. DeltaGf values) upon DNA binding using the SUPREX technique. Ultimately, dissociation constants for each binding interaction were evaluated. An important part of this work was the demonstration that the stoichiometry of protein-DNA complexes could be determined by SUPREX and that the SUPREX technique could be used to probe the folding behavior of DNA binding proteins in dilute solutions.; The results in this work establish the accuracy, precision and generality of the SUPREX technique for quantifying the strength of protein-DNA interactions. SUPREX can be used to differentiate both specific and non-specific binding events. In most cases, the SUPREX technique can be used to accurately predict the Kd value of a protein-DNA complex within a factor of 2 to 5. Important experimental advantages of the SUPREX technique over conventional methods for characterizing the thermodynamic properties of protein-DNA complexes include: the ability to analyze protein samples in complex mixtures and the ability to analyze protein-DNA complexes directly in solution without the attachment of covalent labels to the protein or DNA. Significantly, it is a solution-based methodology and offers a true equilibrium measure for protein-DNA interactions.