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 a-helical GCN4 bZIP by a lanine substitution: 4A, 11A, and 18A contain four, 11, and 18 alanine muta tions in their DNA-binding basic regions, respectively. Titration of fluore scein-labeled duplexes with increasing amounts of protein yielded dissociat ion constants in the low-to-mid nanomolar range for all bZIP mutants in com plex with the AP-1 target site (5 ' -TGACTCA-3 '); binding to the nonspecif ic 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 ura cil-containing AP-1, whereas 11A and 18A bound almost equally well to nativ e AP-1 and uracil-containing AP-1. These modest changes in binding affiniti es may reflect the multivalent nature of protein-DNA interactions, as our h ighly mutated proteins still exhibit native-like behavior. These protein mu tations may compensate for changes in enthalpic and entropic contributions toward DNA-binding in order to maintain binding free energies similar to th at of the native protein-DNA complex. (C) 2001 Elsevier Science Ltd. All ri ghts reserved.