title: A new H/D exchange- and mass spectrometry-based method for thermodynamic analysis of protein-DNA interactions.
abstract: The application of SUPREX (stability of unpurified proteins from rates of H/D exchange) to the thermodynamic analysis of protein-DNA complexes is described. A series of five model protein-DNA complexes involving two known DNA binding proteins, Arc repressor and CopG, were analyzed in order to determine the accuracy, precision, and generality of the SUPREX technique for quantifying the strength of protein-DNA interactions. For protein-DNA complexes that reversibly unfold in a two-state manner, we demonstrate that reasonably precise Kd values in agreement with those determined by conventional techniques can be determined by SUPREX. In the case of protein-DNA complexes that are not well modeled by a two-state unfolding mechanism, we find that relative binding affinities can be determined in the SUPREX experiment.
authors.affiliation: Department of Chemistry, Duke University, Durham, NC 27708, USA.
authors.forename: Liyuan
authors.initials: L
authors.lastname: Ma
authors.forename: Michael C
authors.initials: MC
authors.lastname: Fitzgerald
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D004247:DNA / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D041961:Deuterium Exchange Measurement / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013816:Thermodynamics; D014508:Urea / Q000494:pharmacology
chemical_list: C416526:ArcR protein, bacteria; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D014508:Urea; D004247:DNA