: We previously reported that a basic region/leucine zipper (bZIP) protein, a hybrid of the GCN4 basic region and C/EBP leucine zipper, not only recognizes cognate target sites AP-1 (5′-TGACTCA-3′) and cAMP-response element (CRE) (5′-TGACGTCA-3′) but also binds selectively to noncognate DNA sites: C/EBP (CCAAT/enhancer binding protein, 5′-TTGCGCAA), XRE1 (xenobiotic response element, 5′-TTGCGTGA), HRE (HIF response element, 5′-GCACGTAG), and E-box (5′-CACGTG). In this work, we used electrophoretic mobility shift assay (EMSA) and circular dichroism (CD) for more extensive characterization of the binding of wt bZIP dimer to noncognate sites as well as full-and half-site derivatives, and we examined changes in flanking sequences. Quantitative EMSA titrations were used to measure dissociation constants of this hybrid, wt bZIP, to DNA duplexes: Full-site binding affinities gradually decrease from cognate sites AP-1 and CRE with K d values of 13 and 12 nM, respectively, to noncognate sites with K d values of 120 nM to low µM. DNA-binding selectivity at half sites is maintained; however, half-site binding affinities sharply decrease from the cognate half site (K d ) 84 nM) to noncognate half sites (all K d values > 2 µM). CD shows that comparable levels of R-helical structure are induced in wt bZIP upon binding to cognate AP-1 or noncognate sites. Thus, noncognate sites may contribute to preorganization of stable protein structure before binding target DNA sites. This work demonstrates that the bZIP scaffold may be a powerful tool in the design of small, R-helical proteins with desired DNA recognition properties.In order to examine the relationship between protein structure and DNA-binding function, we exploit the protein R-helix, a structure used ubiquitously for sequence-selective DNA recognition and one that chemists have successfully manipulated in design and synthesis studies for many years (examples include refs 1-8). GCN4 is a dimeric transcriptional regulator that governs histidine biosynthesis in yeast under conditions of amino acid starvation (9). Crystal structures of the basic region/leucine zipper (bZIP) 1 domain of GCN4 bound to the AP-1 site (5′-TGACTCA) (10) and cAMP-response element (CRE) site (5′-TGACGTCA) (11, 12) and the Jun-Fos bZIP heterodimer bound to show that a continuous R-helix provides the basic region interface for binding to specific DNA sites, as well as the leucine zipper coiled coil dimerization structure. The fulllength GCN4 monomer is 281 amino acids, and the bZIP comprises a dimer of ∼60 residue monomers. We previously generated a series of minimalist bZIP proteins comprising alanine-rich variants of the GCN4 basic region fused to the C/EBP leucine zipper: The wt bZIP comprises the native GCN4 basic region. McKnight and co-workers showed that exchanging basic regions and leucine zippers between GCN4 and C/EBP resulted in functional proteins that targeted cognate DNA sites (14), and likewise, we found our wt bZIP hybrid to be functionally equivalent to the GCN4 bZIP (15,16).