Fluorescence spectroscopy and anisotropy in the analysis of DNA-protein interactions.
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.
Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas, Heraklion, Crete, Greece.
Rosy
R
Favicchio
Anatoly I
AI
Dragan
G Geoff
GG
Kneale
Christopher M
CM
Read
D000818:Animals; D020029:Base Pairing; D004247:DNA / Q000378:metabolism*; D005454:Fluorescence Polarization / Q000379:methods*; D024243:HMGB1 Protein / Q000378:metabolism; D007202:Indicators and Reagents; D051379:Mice; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D055755:SOXD Transcription Factors / Q000378:metabolism; D012996:Solutions; D013050:Spectrometry, Fluorescence / Q000379:methods*; D017186:Titrimetry
D024243:HMGB1 Protein; D007202:Indicators and Reagents; D011506:Proteins; D055755:SOXD Transcription Factors; D012996:Solutions; C089186:Sox5 protein, mouse; D004247:DNA