Abstract
Changes in the fluorescence emission spectrum of a protein upon binding to DNA can often be used to determine the stoichiometry of binding and equilibrium binding constants; in some cases the data can also give an indication of the location of particular residues within the protein. The experiments are generally quick and easy to perform, requiring only small quantities of material (1). Spectroscopic techniques allow one to measure binding at equilibrium (unlike, for example, gel retardation assays and other separation techniques that are strictly nonequilibrium methods). Fluorescence is one of the most sensitive of spectroscopic techniques, allowing the low concentrations (typically in the nanomolar to micromolar range) required for estimation of binding constants for many protein-DNA interactions. Considerable care, however, needs to be exercised in the experiment itself and in the interpretation of results. The fundamental principles of fluorescence are discussed briefly in the remainder of the Introduction.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Harris, D. A. and Bashford, C. L. (eds.) (1987) Spectrophotometry and Spectrofluorimetry: A Practical Approach. IRL Press, Oxford, UK. (Chapters 1 and 4 are particularly relevant.)
Kneale G. G. and Wijnaendts van Resandt, R. W. (1985) Time resolved fluorescence of the Pf1 bacteriophage DNA-binding protein: determination of oligo-and polynucleotide binding parameters. Eur. J. Biochem. 149, 85–93.
Birdsall, B., King, R. W., Wheeler, M. R., Lewis, C. A., Goode, S. R., Dunlap, R. B. et al. (1983) Anal. Biochem. 132, 353–361.
Carpenter, M. L. and Kneale, G. G. (1991) Circular dichroism and fluorescence analysis of the interaction of Pf1 gene 5 protein with poly(dT). J. Mol. Biol. 217, 681–689.
Greulich, K. O., Wijnaendts van Resandt, R. W., and Kneale G. G (1985) Time resolved fluorescence of bacteriophage Pf1 DNA-binding protein and its complex with DNA. Eur. Biophys. J. 11, 195–201.
Mély, Y., de Rocquigny, H., Sorinas-Jimeno, M., Keith, G., Roques, B. P., Marquet, R., et al. (1995) Binding of the HIV-1 nucleocapsid protein to the primer tRNA3 Lys in vitro, is essentially not specific. J. Biol. Chem. 270, 1650–1656.
Kim, C. and Wold, M. S. (1995) Recombinant human replication protein A binds to polynucleotides with low cooperativity. Biochemistry 34, 2058–2064.
Soengas, M. S., Mateo, C. R., Salas, M., Acuña, A. U., and Gutiérrez, C. (1997) Structural features of ф 29 single-stranded DNA-binding protein. J. Biol. Chem. 272, 295–302.
Kelly, R. C., Jensen, D. E., and von Hippel, P. H. (1976) Fluorescence measurements of binding parameters for bacteriophage T4 gene 32 protein to mono-, oligo-, and polynucleotides. J. Biol. Chem. 251, 7240–7250.
McGhee, J. D. and von Hippel, P. H. (1974) Theoretical aspects of DNA-protein interactions: cooperative and non-cooperative binding of large ligands to a one-dimensional homogeneous lattice. J. Mol. Biol. 86, 469–489.
Alma, N. C. M., Harmsen, B. J. M., de Jong, E. A. M., Ven, J. V. D., and Hilbers, C. W. (1983) Fluorescence studies of the complex formation between the gene 5 protein of bacteriophage M13 and polynucleotides. J. Mol. Biol. 163, 47–62.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2001 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Carpenter, M.L., Oliver, A.W., Geoff Kneale, G. (2001). Analysis of DNA-Protein Interactions by Intrinsic Fluorescence. In: Moss, T. (eds) DNA-Protein Interactions. Methods in Molecular Biology, vol 148. Humana Press. https://doi.org/10.1385/1-59259-208-2:491
Download citation
DOI: https://doi.org/10.1385/1-59259-208-2:491
Publisher Name: Humana Press
Print ISBN: 978-0-89603-625-3
Online ISBN: 978-1-59259-208-1
eBook Packages: Springer Protocols