A general method of analysis of ligand binding to competing macromolecules. Application to the E. coli DnaB helicase-nucleic acid interactions

M. J. Jezewska, W. Bujalowski

    Research output: Contribution to journalArticlepeer-review

    Abstract

    Quantitative analyses of protein-nucleic acid interactions in solution are greatly facilitated if the formation of the complex is accompanied by a large spectroscopic signal change. However, there are many instances when protein-nucleic acid interactions do not induce adequate changes in their spectroscopic properties. We describe a general method to analyze such interactions. The method is based on titrations of a reference nucleic acid, with the protein in the presence of a competing nucleic acid, whose interaction parameters with the protein are to be determined. The Macromolecule Competition Titration (MCT) method allows for the determination of the absolute average binding density, unavailable by other methods, and construction of a modelindependent true isotherm. To analyze binding of a ligand to competing nucleic acids, we introduced the combined application of the McGhee-von Hippel and the Epstein theories. Our approach allows a direct fit of the entire binding isotherm without resorting to numerical calculations. Using the MCT method in conjuction with photo-cross-linking techniques we obtained quantitative and structural characterization of interactions of E. coli primary replicative helicase, DnaB protein with ssDNA. (Supported by NIH Grant 46679 and John Sealy Grant 2545/95).

    Original languageEnglish (US)
    Pages (from-to)A1190
    JournalFASEB Journal
    Volume11
    Issue number9
    StatePublished - Dec 1 1997

    ASJC Scopus subject areas

    • Biotechnology
    • Biochemistry
    • Molecular Biology
    • Genetics

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