Relative DNA binding affinity of helix 3 homeodomain analogues, major groove binders, can be rapidly screened by displacement of prebound ethidium bromide. A comparative study.
The binding affinity for a 12-bp dsDNA of Antennapedia helix 3 analogues, major groove binders, has been measured by displacement of prebound ethidium bromide, a fluorescent displacement assay proposed for minor groove binders by Boger et al.(J. Am. Chem. Soc., 2000, 122, 6382-6394). Relative binding affinities determined by this method were compared to those obtained by gel mobility shift and footprinting assays for the 12-bp dsDNA and a 178-bp DNA fragment. The present work demonstrates that the fluorescence displacement assay is suitable for rapid screening of major groove binders, even though about 60 to 70% of the prebound ethidium bromide is displaced by these peptides. Total (100%) displacement of ethidium bromide was serendipitously achieved by addition in the peptide sequence, at the N-terminus, of a S-3-nitro-2-pyridinesulfenyl-N-acetyl-cysteine residue. S-3-nitro-2-pyridinesulfenylcysteine was shown to (i) bind to dsDNA with a micromolar affinity and (ii) direct within DNA grooves a peptide with no affinity for dsDNA.
UMR 7613 CNRS - Paris VI, Case 182, Université P. et M. Curie, 4, Place Jussieu, 75005 Paris, France.
Yong-Ho
YH
Shim
Paola B
PB
Arimondo
Alain
A
Laigle
Anna
A
Garbesi
Solange
S
Lavielle
D000595:Amino Acid Sequence; D051943:Antennapedia Homeodomain Protein; D001483:Base Sequence; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D004996:Ethidium / Q000737:chemistry*; D018398:Homeodomain Proteins / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism
D051943:Antennapedia Homeodomain Protein; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; D010455:Peptides; D014157:Transcription Factors; D004247:DNA; D004996:Ethidium; D003545:Cysteine