The effect of C-terminal helix stabilization on specific DNA binding by monomeric GCN4 peptides.
DNA binding by a 29-residue, monomeric, GCN4 basic region peptide, GCN4br, as well as by peptide br-C, a monomeric basic-region analogue that is helix stabilized at its C-terminal end by a Lys25. Asp29 side-chain lactam-bridged alanine-rich sequence, was studied at 25 C in an aqueous buffer containing 100 mm NaCl. Mixing of both peptides with duplex DNA containing the cAMP-responsive element (CRE) was accompanied by significant helix stabilization in the peptides, whereas mixing of the peptides with duplex DNA containing a scrambled CRE site was not. Peptide NBD-br-C was synthesized as a fluorescent probe to evaluate these peptide-DNA interactions further. Quantitative analysis of the fluorescence quenching of peptide NBD-br-C by CRE half-site DNA indicated the formation of a 1:1 complex with a dissociation constant of 1.41 +/- 0.22 microm. Competitive displacement fluorescence assays of CRE half-site binding gave dissociation constants of 0.65 +/- 0.09 microm for peptide br-C and 3.9 +/- 0.5 microM for GCN4br, which corresponds to a free energy difference of 1.1 kcal/mol that is attributed to the helix stabilization achieved in peptide br-C. This result indicates that helix initiation by the alpha-helical leucine zipper dimerization motif in native bzip proteins, such as GCN4, contributes significantly to the affinity of basic region peptides for their recognition sites on DNA. Our fluorescence assay should also prove useful for determining dissociation constants for CRE binding by other GCN4 basic region analogues under equilibrium conditions and physiological salt concentrations.
Zhang
Min
M
Rutgers University, Department of Chemistry and Chemical Biology, Piscataway, NJ 08854, USA.
Wu
Bing
B
Zhao
Hong
H
Taylo
John W
JW
D002942:Circular Dichroism; D004268:DNA-Binding Proteins; D004305:Dose-Response Relationship, Drug; D005456:Fluorescent Dyes / Q000494:pharmacology; D007700:Kinetics; D008856:Microscopy, Fluorescence; D008956:Models, Chemical; D009841:Oligonucleotides / Q000494:pharmacology; D010452:Peptide Biosynthesis; D010455:Peptides / Q000737:chemistry*; D011485:Protein Binding; D011494:Protein Kinases / Q000737:chemistry* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism*
D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D009841:Oligonucleotides; D010455:Peptides; D029701:Saccharomyces cerevisiae Proteins; D011494:Protein Kinases