Quantitative distance information on protein-DNA complexes determined in polyacrylamide gels by fluorescence resonance energy transfer.
In polyacrylamide gels, we have quantitatively determined Forster transfer (fluorescense resonance energy transfer, FRET) between two fluorescent dyes attached to DNA in protein-DNA complexes. The donor-dye fluorescein labeled to DNA retains its free mobility in the polyacrylamide gel, however, its fluorescence properties change. The different quantum yield of fluorescein in the gel is found to be independent of the gel concentration and can thus be quantitatively taken into account by a reduced Forster distance R0 of 46 A compared to 50 A in solution. We have determined global structural properties of two proteins binding to double-labeled DNA using a novel gel-based fluorescence resonance energy transfer assay. In polyacrylamide gels we have analyzed the binding of integration host factor (IHF) and the high mobility group protein NHP6a to their substrate DNA. The measured Forster transfer efficiency allows us to deduce information on the overall shape and the DNA bending angle in the complex.
Lorenz
M
M
Department of Molecular Biology, Institute for Molecular Biotechnology, Jena, Germany.
Diekmann
S
S
D000180:Acrylic Resins; D001426:Bacterial Proteins / Q000737:chemistry*; D004247:DNA / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D019793:Fluorescein; D005454:Fluorescence Polarization / Q000379:methods; D024001:HMGN Proteins; D006609:High Mobility Group Proteins / Q000737:chemistry*; D034982:Integration Host Factors; D009687:Nuclear Proteins / Q000737:chemistry*; D011485:Protein Binding; D029701:Saccharomyces cerevisiae Proteins; D013050:Spectrometry, Fluorescence
D000180:Acrylic Resins; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D024001:HMGN Proteins; D006609:High Mobility Group Proteins; D034982:Integration Host Factors; C086471:NHP6A protein, S cerevisiae; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; C016680:polyacrylamide gels; D004247:DNA; D019793:Fluorescein