Photocontrol of DNA binding specificity of a miniature engrailed homeodomain.
Control of DNA binding of HDH-3, a 18-residue polypeptide based on the recognition helix of the Q50K engrailed homeodomain, has been achieved. HDH-3 was linked to an azobenzene cross-linker through two cysteine residues in an i, i + 11 spacing. For the thermodynamically stable trans configuration of the cross-linker, the dark-adapted peptide (dad-HDH-3) adopted a mainly alpha-helical structure as judged by circular dichroism (CD) spectroscopy. After irradiation with light of 360 nm, the helical content of the peptide (irrad-HDH-3) was reduced significantly and the CD spectrum of the irradiated peptide resembled that of the largely unstructured, unalkylated peptide. Despite lacking helices-1 and -2 and the N-terminal arm of Q50K engrailed, dad-HDH-3 bound to its natural DNA target sequence TAATCC (QRE) with high affinity (K(D) = 7.5 +/- 1.3 nM). The binding affinity for the mutant DNA sequence, TAATTA (ERE), was reduced significantly (K(D) = 140 +/- 11 nM). Unlike irrad-HDH-3, which like the unalkylated parent peptide displayed only marginal DNA binding specificity, dad-HDH-3 specified base pairs 5 and 6 of QRE with an accuracy rivaling that of the intact wild-type Q50K engrailed homeodomain, making dad-HDH-3 the most specific designed DNA binding miniature homeodomain reported to date. Moreover, DNA binding affinity and specificity of HDH-3 could be controlled externally by irradiation with light.
Guerrero
Lucia
L
School of Chemistry, Cardiff University, Main Building, Park Place, Cardiff CF10 3AT, Wales, U.K.
Smart
Oliver S
OS
Woolley
G Andrew
GA
Allemann
Rudolf K
RK
D001391:Azo Compounds; D002942:Circular Dichroism; D003432:Cross-Linking Reagents; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry; D005454:Fluorescence Polarization; D018398:Homeodomain Proteins / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry*; D010777:Photochemistry
D001391:Azo Compounds; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D010455:Peptides; D004247:DNA; C009850:azobenzene