Quantifying DNA-Protein interactions by single molecule stretching

被引：2
作者：
Williams, Mark C.
Rouzina, Ioulia
Karpel, Richard L.
机构：
[1] Northeastern Univ, Dept Phys, Boston, MA 02115 USA
[2] Northeastern Univ, Ctr Interdisciplinary Res Complex Syst, Boston, MA 02115 USA
[3] Univ Minnesota, Dept Biochem Mol Biol & Biophys, Minneapolis, MN 55455 USA
[4] Univ Maryland Baltimore Cty, Dept Chem & Biochem, Baltimore, MD 21250 USA
来源：
BIOPHYSICAL TOOLS FOR BIOLOGISTS: VOL 1 IN VITRO TECHNIQUES | 2008年 / 84卷
基金：
美国国家科学基金会;
关键词：
D O I：
10.1016/S0091-679X(07)84017-9
中图分类号：
Q2 [细胞生物学];
学科分类号：
071009 ; 090102 ;
摘要：
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.
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页码：517 / +
页数：25
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