title: Quantifying DNA-protein interactions by single molecule stretching.
abstract: In this chapter, we discuss a new method for quantifying DNA-protein interactions. A single double-stranded DNA (dsDNA) molecule is stretched beyond its contour length, causing the base pairs to break while increasing the length from that of dsDNA to that of ssDNA. When applied in a solution containing DNA binding ligands, this method of force-induced DNA melting can be used to quantify the free energy of ligand binding, including the free energy of protein binding. The dependence of melting force on protein concentration is used to obtain the equilibrium binding constant of the ligand to DNA. We have applied this method to a well-studied DNA-binding protein, bacteriophage T4 gene 32 protein (gp32), and have obtained binding constants for the protein to single-stranded DNA (ssDNA) under a wide range of solution conditions. Our analysis of measurements conducted at several salt concentrations near physiological conditions indicates that a salt-dependent conformational change regulates DNA binding by gp32.
authors.affiliation: Department of Physics, 111 Dana Research Center, Northeastern University, Boston, Massachusetts 02115, USA.
authors.forename: Mark C
authors.initials: MC
authors.lastname: Williams
authors.forename: Ioulia
authors.initials: I
authors.lastname: Rouzina
authors.forename: Richard L
authors.initials: RL
authors.lastname: Karpel
mesh_terms: D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008954:Models, Biological; D009691:Nucleic Acid Denaturation / Q000187:drug effects; D052898:Optical Tweezers; D011485:Protein Binding / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D017434:Protein Structure, Tertiary; D012965:Sodium Chloride / Q000494:pharmacology; D055672:Static Electricity; D013816:Thermodynamics; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; D014764:Viral Proteins; C060035:gp32 protein, Enterobacteria phage T4; D012965:Sodium Chloride; D004247:DNA