The contribution of the methyl groups on thymine bases to binding specificity and affinity by alanine-rich mutants of the bZIP motif

• Published in: Bioorganic & medicinal chemistry 2001-09, Vol.9 (9), p.2485-2491
• Main Authors: Kise, Kenneth J., Shin, Jumi A.
• Format: Article
• Language: English
• Subjects: Alanine - chemistry; Amino Acid Motifs; Amino Acid Substitution; Basic-Leucine Zipper Transcription Factors; Binding Sites; Biological and medical sciences; DNA - metabolism; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Fluorescence Polarization; Fundamental and applied biological sciences. Psychology; G-Box Binding Factors; Molecular and cellular biology; Molecular genetics; Mutation; Protein Binding; Thermodynamics; Thymine - chemistry; Titrimetry; Transcription Factors - chemistry; Transcription Factors - genetics; Transcription Factors - metabolism; Transcription. Transcription factor. Splicing. Rna processing; Uracil - chemistry; Yeasts - chemistry
• ISSN: 0968-0896; 1464-3391
• DOI: 10.1016/S0968-0896(01)00216-4

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 α-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