Quantifying DNA-Protein Interactions by Single Molecule Stretching

Mark C. Williams, Ioulia Rouzina, Richard L. Karpel

Research output: Chapter in Book/Report/Conference proceedingChapter

3 Scopus citations


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.

Original languageEnglish (US)
Title of host publicationBiophysical Tools for Biologists, Volume One
Subtitle of host publicationIn Vitro Techniques
EditorsJohn Correi, William Detrich, III
Number of pages24
StatePublished - 2008

Publication series

NameMethods in Cell Biology
ISSN (Print)0091-679X

Bibliographical note

Funding Information:
The authors wish to thank Kiran Pant for her work on the development of the method of DNA force‐induced melting in the presence of gp32 and Micah McCauley for the original drawing of Fig. 1 . Funding for this work was provided by NIH (GM 52049, RLK and GM 72462, MCW) and NSF (MCB‐0238190, MCW).

Copyright 2009 Elsevier B.V., All rights reserved.


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