Summary
Fluorescence spectroscopy can be used as a sensitive non-destructive technique for the characterisation of protein–DNA interactions. A comparison of the intrinsic emission spectra obtained for a protein–DNA complex and for free protein can be informative about the environment of tryptophan and tyrosine residues in the two states. Often there is quenching of the fluorescence intensity of an intrinsic emission spectrum and/or a shift in the wavelength maximum on protein binding to DNA. A step-by-step protocol describes the determination of a DNA-binding curve by measurement of the quenching of the intrinsic protein fluorescence.
Fluorescence anisotropy can also be used to obtain a DNA-binding curve if the molecular size of the protein–DNA complex is sufficiently different from the free fluorescing component. Typically an extrinsic fluorophore attached to one or both 5′ ends of single-stranded or duplex DNA is used, for this increases the sensitivity of measurement.
Fitting of the binding curves, assuming a model, can often yield the stoichiometry and association constant of the interaction. The approach is illustrated using the interaction of the DNA-binding domains (HMG boxes) of mouse Sox-5 and mammalian HMGB1 with short DNA duplexes.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Harris, D.A. and Bashford, C.L., eds. (1987). Spectrophotometry and Spectrofluorimetry: A Practical Approach. IRL Press, Oxford, England (Chaps. 1 and 4 are particularly relevant).
Lakowicz, J.R. (2006). Principles of Fluorescence Spectroscopy. 3rd edn. Springer, New York, USA.
Clegg, R.M., Murchie, A.I. and Lilley, D.M. (1994). The solution structure of the four-way DNA junction at low-salt conditions: a fluorescence resonance energy transfer analysis. Biophys. J. 66, 99–109.
Eis, P.S. and Millar, D.P. (1993). Conformational distributions of a four-way DNA junction revealed by time-resolved fluorescence resonance energy transfer. Biochemistry. 32, 13852–13860.
Phillips, N.B., Jancso-Radek, A., Ittah, V., Singh, R., Chan, G., Haas, E. and Weiss, M.A. (2006). SRY and human sex determination: the basic tail of the HMG box functions as a kinetic clamp to augment DNA bending. J. Mol. Biol. 358, 172–192.
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. Jr., Goode, S.R., Dunlap, R.B. and Roberts, G.C. (1983). Correction for light absorption in fluorescence studies of protein-ligand interactions. Anal. Biochem. 132, 353–361.
Mély, Y., de Rocquigny, H., Sorinas-Jimeno, M., Keith, G., Roques, B.P., Marquet, R. and Gérard, D. (1995). Binding of the HIV-1 nucleocapsid protein to the primer tRNA3Lys 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.
Privalov, P.L., Jelesarov, I., Read, C.M., Dragan, A.I. and Crane-Robinson, C. (1999). The energetics of HMG box interactions with DNA. Thermodynamics of the DNA binding of the HMG box from mouse Sox-5. J. Mol. Biol. 294, 997–1013.
Dragan, A.I., Liggins, J.R., Crane-Robinson, C. and Privalov, P.L. (2003). The energetics of specific binding of AT-hooks from HMGA1 to target DNA. J. Mol. Biol. 327, 393–411.
Dragan, A.I., Frank, L., Liu, Y., Makeyeva, E.N., Crane-Robinson, C. and Privalov, P.L. (2004). Thermodynamic signature of GCN4-bZIP binding to DNA indicates the role of water in discriminating between the AP-1 and ATF/CREB sites. J. Mol. Biol. 343, 865–878.
Hargreaves, V.V., Makeyeva, E.N., Dragan, A.I. and Privalov, P.L. (2005). Stability and DNA binding ability of the DNA binding domains of interferon regulatory factors 1 and 3. Biochemistry. 44, 14202–14209.
Dragan, A.I., Li, Z., Makeyeva, E.N., Milgotina, E.I., Liu, Y., Crane-Robinson, C. and Privalov, P.L. et al (2006). Forces driving the binding of homeodomains to DNA. Biochemistry. 45, 141–51.
Dragan, A.I., Klass, J., Read, C., Churchill, M.E., Crane-Robinson, C. and Privalov, P.L. (2003). DNA binding of a non-sequence-specific HMG-D protein is entropy driven with a substantial non-electrostatic contribution. J. Mol. Biol. 331, 795–813.
Dragan, A.I., Read, C.M., Makeyeva, E.N., Milgotina, E.I., Churchill, M.E., Crane-Robinson, C. and Privalov, P.L. (2004). DNA binding and bending by HMG boxes: energetic determinants of specificity. J. Mol. Biol. 343, 371–393.
Dragan, A.I., Liu, Y., Makeyeva, E.N. and Privalov, P.L. (2004). DNA-binding domain of GCN4 induces bending of both the ATF/CREB and AP-1 binding sites of DNA. Nucleic Acids Res. 32, 5192–5197.
Denny, P., Swift, S., Connor, F. and Ashworth, A. (1992). An Sry-related gene expressed during spermatogenesis in the mouse encodes a sequence-specific DNA-binding protein. EMBO J. 11, 3705–3712.
Ellwood, K.B., Yen, Y.M., Johnson, R.C. and Carey, M. (2000). Mechanism for specificity by HMG-1 in enhanceosome assembly. Mol. Cell. Biol. 20, 4359–4370.
Read, C.M., Cary, P.D., Preston, N.S., Lnenicek-Allen, M. and Crane-Robinson, C. (1994). The DNA sequence specificity of HMG boxes lies in the minor wing of the structure. EMBO J. 13, 5639–5646.
Teo, S.H., Grasser, K.D. and Thomas, J.O. (1995). Differences in the DNA binding properties of the HMG-box domains of HMG1 and the sex-determining factor SRY. Eur. J. Biochem. 230, 943–950.
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.
Crane-Robinson, C., Read, C.M., Cary, P.D., Driscoll, P.C., Dragan, A.I. and Privalov, P.L. (1998). The energetics of HMG box interactions with DNA. Thermodynamic description of the box from mouse Sox-5. J. Mol. Biol. 281, 705–717.
Behlke, M.A., Huang, L., Bogh, L., Rose, S. and Devor, E.J. (2005). Fluorescence quenching by proximal G-bases. Integrated DNA Technologies Research Report. http://www.idtdna.com.
Bogh, L.D. and Behlke, M.A. (2005). Spectra of DNA-conjugated fluorescent dyes. Integrated DNA Technologies Research Report. http://www.idtdna.com.
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.
Acknowledgements
We gratefully acknowledge the financial support from a Wellcome Trust grant to the Portsmouth laboratory and an NSF grant (MCB 0519381) to the Baltimore laboratory. R.F. would like to thank IBBS, University of Portsmouth for a bursary.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Favicchio, R., Dragan, A.I., Kneale, G.G., Read, C.M. (2009). Fluorescence Spectroscopy and Anisotropy in the Analysis of DNA-Protein Interactions. In: Leblanc, B., Moss, T. (eds) DNA-Protein Interactions. Methods in Molecular Biology™, vol 543. Humana Press. https://doi.org/10.1007/978-1-60327-015-1_35
Download citation
DOI: https://doi.org/10.1007/978-1-60327-015-1_35
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-60327-014-4
Online ISBN: 978-1-60327-015-1
eBook Packages: Springer Protocols