The contribution of the methyl groups on thymine bases to binding specificity and affinity by alanine-rich mutants of the bZIP motif.
We have used fluorescence anisotropy to measure in situ the thermodynamics of binding of alanine-rich mutants of the GCN4 basic region/leucine zipper (bZIP) to short DNA duplexes, in which thymines were replaced with uracils, in order to quantify the contributions of the C5 methyl group on thymines with alanine methyl side chains. We simplified the alpha-helical GCN4 bZIP by alanine substitution: 4A, 11A, and 18A contain four, 11, and 18 alanine mutations in their DNA-binding basic regions, respectively. Titration of fluorescein-labeled duplexes with increasing amounts of protein yielded dissociation constants in the low-to-mid nanomolar range for all bZIP mutants in complex with the AP-1 target site (5'-TGACTCA-3'); binding to the nonspecific control duplex was >1000-fold weaker. Small changes of <1 kcal/mol in binding free energies were observed for wild-type bZIP and 4A mutant to uracil-containing AP-1, whereas 11A and 18A bound almost equally well to native AP-1 and uracil-containing AP-1. These modest changes in binding affinities may reflect the multivalent nature of protein-DNA interactions, as our highly mutated proteins still exhibit native-like behavior. These protein mutations may compensate for changes in enthalpic and entropic contributions toward DNA-binding in order to maintain binding free energies similar to that of the native protein-DNA complex.
Kise
K J
KJ
Department of Chemistry, University of Pittsburgh, Pittsburgh, PA 15260, USA.
Shin
J A
JA
D000409:Alanine / Q000737:chemistry*; D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D050976:Basic-Leucine Zipper Transcription Factors; D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005454:Fluorescence Polarization; D051790:G-Box Binding Factors; D009154:Mutation; D011485:Protein Binding; D013816:Thermodynamics; D013941:Thymine / Q000737:chemistry*; D017186:Titrimetry; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014498:Uracil / Q000737:chemistry; D015003:Yeasts / Q000737:chemistry
D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; D051790:G-Box Binding Factors; D014157:Transcription Factors; D014498:Uracil; D004247:DNA; D000409:Alanine; D013941:Thymine