title: Analytical Ultracentrifugation as a Tool to Study Nonspecific Protein-DNA Interactions.
abstract: Analytical ultracentrifugation (AUC) is a powerful tool that can provide thermodynamic information on associating systems. Here, we discuss how to use the two fundamental AUC applications, sedimentation velocity (SV), and sedimentation equilibrium (SE), to study nonspecific protein-nucleic acid interactions, with a special emphasis on how to analyze the experimental data to extract thermodynamic information. We discuss three specific applications of this approach: (i) determination of nonspecific binding stoichiometry of E. coli integration host factor protein to dsDNA, (ii) characterization of nonspecific binding properties of Adenoviral IVa2 protein to dsDNA using SE-AUC, and (iii) analysis of the competition between specific and nonspecific DNA-binding interactions observed for E. coli integration host factor protein assembly on dsDNA. These approaches provide powerful tools that allow thermodynamic interrogation and thus a mechanistic understanding of how proteins bind nucleic acids by both specific and nonspecific interactions.
authors.affiliation: Pfizer, Pharmaceutical Research and Development, Chesterfield, Missouri, USA.
authors.forename: Teng-Chieh
authors.initials: TC
authors.lastname: Yang
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado, Anschutz Medical Campus, Aurora, Colorado, USA.
authors.forename: Carlos Enrique
authors.initials: CE
authors.lastname: Catalano
authors.affiliation: Alliance Protein Laboratories, San Diego, California, USA. Electronic address: karl.maluf@ap-lab.com.
authors.forename: Nasib Karl
authors.initials: NK
authors.lastname: Maluf
mesh_terms: D000465:Algorithms; D001667:Binding, Competitive; D004247:DNA / Q000737:chemistry* / Q000302:isolation & purification; D008958:Models, Molecular; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification; D013816:Thermodynamics; D014461:Ultracentrifugation
chemical_list: D011506:Proteins; D004247:DNA
keywords: Nonspecific binding; Specific and nonspecific competitive binding