Combining a Base Deaminase Mutator with Phage-Assisted Evolution.

Phage-assisted evolution has emerged as a powerful technique for improving a protein's function by using mutagenesis and selective pressure. However, mutations typically occur throughout the host's genome and are not limited to the gene-of-interest (GOI): these undesirable genomic mutations can yield host cells that circumvent the system's selective pressure. Our system targets mutations specifically toward the GOI by combining T7 targeted mutagenesis and phage-assisted evolution. This system improves the structure and function of proteins by accumulating favorable mutations that can change its binding affinity, specificity, and activity.

Combining Rational Design and Continuous Evolution on Minimalist Proteins That Target the E-box DNA Site.

Protein-based therapeutics are part of the next-generation arsenal of drugs being developed against proto-oncoprotein Myc. We designed protein MEF to mimic the basic region/helix-loop-helix/leucine zipper (bHLHZ) domain of Max and Myc, which bind to the E-box motif (enhancer box, CACGTG). To make MEF, we started with our rationally designed ME47, a hybrid of the Max basic region and E47 HLH, that effectively inhibited tumor growth in a mouse model of breast cancer. We used phage-assisted continuous evolution (PACE), which uncovered mutations at Arg12 that contact the DNA phosphodiester backbone. The Arg12 mutations improved ME47's stability. We replaced Cys29 with Ala to eliminate potential undesired disulfide formation and fused the designed FosW leucine zipper to mutated ME47 to increase the dimerization interface and E-box targeting activity. This "franken-protein" MEF comprises the Max basic region, E47 HLH, and FosW leucine zipper. Compared with ME47, MEF gives 2-fold stronger binding to E-box and 4-fold increased specificity for E-box over nonspecific DNA. The synergistic combination of rational design and PACE allowed us to make MEF and demonstrates the power and utility of our two-pronged approach toward development of promising protein drugs with robust structure and DNA-binding function.

Phage-Assisted Continuous Evolution (PACE): A Guide Focused on Evolving Protein-DNA Interactions.

The uptake of directed evolution methods is increasing, as these powerful systems can be utilized to develop new biomolecules with altered/novel activities, for example, proteins with new catalytic functions or substrate specificities and nucleic acids that recognize an intended target. Especially useful are systems that incorporate continuous evolution, where the protein under selective pressure undergoes continuous mutagenesis with little-to-no input from the researcher once the system is started. However, continuous evolution methods can be challenging to implement and a daunting investment of time and resources. Our intent is to provide basic information and helpful suggestions that we have gained from our experience with bacterial phage-assisted continuous evolution (PACE) toward the evolution of proteins that bind to a specific DNA target. We discuss factors to consider before adopting PACE for a given evolution scheme with focus on the PACE bacterial one-hybrid selection system and what optimization of a PACE selection circuit may look like using the evolution of the DNA-binding protein ME47 as a case study. We outline different types of selection circuits and techniques that may be added onto a basic PACE setup. With this information, researchers will be better equipped to determine whether PACE is a valid strategy to adopt for their research program and how to set up a valid selection circuit.

The Intrinsically Disordered Loop in the USF1 bHLHZ Domain Modulates Its DNA-Binding Sequence Specificity in Hereditary Asthma.

USF1, a basic region/helix-loop-helix/leucine zipper (bHLHZ) transcription factor, binds to the E-box in the PAI-1 (plasminogen activator inhibitor) promoter. Two alleles containing the E-box control PAI-1 transcription; these alleles are termed "4G" and "5G" based on the G tract flanking E-box. USF1-governed transcription of PAI-1 is elevated in heritable asthma sufferers: the 4G/4G genotype has the highest plasma levels of PAI-1. While USF1 uses its basic region to bind E-box, we found that it uses its 12 amino-acid loop to recognize the flanking sequence and discern the single-nucleotide difference between the alleles. We used the bacterial one-hybrid and electrophoretic mobility shift assays to assess protein-DNA recognition, and circular dichroism to examine protein secondary structure. We mutated Ser233 and Thr234 in the USF1 bHLHZ loop to Ala to generate S233A and T234A. Interestingly, USF1 bHLHZ, S233A, and T234A prefer the 5G sequence (USF1 bHLHZ Kd values 4.1 ± 0.3 nM and 7.0 ± 0.4 nM for 5G and 4G, respectively), whereas studies in stimulated human mast cells showed a preference for 4G. We replaced the 8 amino-acid loop of transcription factor Max bHLHZ with the 12 amino-acid USF1 loop: this mutant now distinguishes the 4G/5G polymorphism-while Max bHLHZ does not-confirming that USF1 differentiation of the 4G/5G is driven by the loop.

Yeast Populations Evolve to Resist CdSe Quantum Dot Toxicity.

Engineered nanomaterials are used globally in biomedical, electronic, and optical devices, and are often discarded into the environment. Cell culture experiments have shown that many inorganic nanoparticles are toxic to eukaryotic cells. Here, we show that populations of eukaryotic cells can evolve to survive chronic exposure to toxic CdSe semiconductor quantum dots (QDs). We grew yeast Saccharomyces cerevisiae for 24 days in liquid medium containing QDs prepared daily at half the minimum inhibitory concentration (MIC50) of the progenitor yeast cells. After 24 days, the cells grew normally under constant exposure to QDs. We concluded that these cells evolved to resist QD toxicity. Surprisingly, when we removed QDs from the growth medium, some of the evolved cells grew poorly, i.e., they grew better in the presence of QDs. Finally, genetic analysis confirmed that the ubiquitin ligase gene bul1 was mutated in the evolved cells, which suggests that this gene may be implicated in increased CdSe QD tolerance. This study shows that chronic exposure to QDs can exert selective pressure causing irreversible genetic changes leading to adaptation.

The DNA target determines the dimerization partner selected by bHLHZ-like hybrid proteins AhRJun and ArntFos.

The molecular basis of protein-partner selection and DNA binding of the basic helix-loop-helix (bHLH) and basic region-leucine zipper (bZIP) superfamilies of dimeric transcription factors is fundamental toward understanding gene regulation. Because these families share structural similarities, we swapped the bHLH and leucine zipper (LZ) modules between families to uncover how individual modules influence protein-partnering and protein:DNA complexation. We previously described ArntFos, a bHLHZ-like hybrid of the bHLH domain from the bHLH/PAS protein Arnt and LZ from the bZIP protein c-Fos, binding to the Arnt E-box site (TCACGTGA) as a homodimer. Herein, we describe a heterodimer between ArntFos and AhRJun, a hybrid of the bHLH domain from AhR and LZ of JunD. We designed AhRJun and ArntFos to heterodimerize, given the strong interaction between native AhR/Arnt and Jun/Fos, but the hybrids showed no preference for hetero- or homo-dimerization in Y2H assays. However, adding a specific DNA target drove the formation of a single dimeric protein species over others. EMSA showed that the AhRJun/ArntFos heterodimer binds to the cognate DNA site XRE1 (TTGCGTG) with Kd = 337 nM. Unexpectedly, the palindromic Arnt E-box drove the binding of the AhRJun/ArntFos heterodimer (Kd = 276 nM)-not the ArntFos homodimer-that binds to the Arnt E-box. However, the dimerization preference switched to the ArntFos homodimer when the variant Max E-box (CCACGTGG) was used. We conclude that the DNA sites themselves are the primary determinants of dimerization specificity for AhRJun and ArntFos, not the JunD and c-Fos LZs, a result that sheds light on the dynamics of protein/protein and protein:DNA interactions and the structural modularity of bHLH and bZIP proteins.

The role of ligand density and size in mediating quantum dot nuclear transport.

Studying the effects of the physicochemical properties of nanomaterials on cellular uptake, toxicity, and exocytosis can provide the foundation for designing safer and more effective nanoparticles for clinical applications. However, an understanding of the effects of these properties on subcellular transport, accumulation, and distribution remains limited. The present study investigates the effects of surface density and particle size of semiconductor quantum dots on cellular uptake as well as nuclear transport kinetics, retention, and accumulation. The current work illustrates that cellular uptake and nuclear accumulation of nanoparticles depend on surface density of the nuclear localization signal (NLS) peptides with nuclear transport reaching a plateau at 20% surface NLS density in as little as 30 min. These intracellular nanoparticles have no effects on cell viability up to 72 h post treatment. These findings will set a foundation for engineering more sophisticated nanoparticle systems for imaging and manipulating genetic targets in the nucleus.

Boundaries of the origin of replication: creation of a pET-28a-derived vector with p15A copy control allowing compatible coexistence with pET vectors.

During our studies involving protein-DNA interactions, we constructed plasmid pSAM to fulfill two requirements: 1) to facilitate transfer of cloned sequences from widely used expression vector pET-28a(+), and 2) to provide a vector compatible with pBR322-derived plasmids for use in cells harboring two different plasmids. Vector pSAM is a pET-28a(+)-derived plasmid with the p15A origin of replication (ori); pET-28a(+) contains the pBR322 replicon that is incompatible with other pBR322-derived plasmids. By replacing the original pET-28a(+) replicon-comprising the ori, RNAI, RNAII, and Rom-with the p15A replicon, we generated pSAM, which contains the pET-28a(+) multiple cloning site and is now compatible with pBR322-derived vectors. Plasmid copy number was assessed using quantitative PCR: pSAM copy number was maintained at 18±4 copies per cell, consistent with that of other p15A-type vectors. Compatibility with pBR322-derived vectors was tested with pGEX-6p-1 and pSAM, which maintained their copy numbers of 49±10 and 14±4, respectively, when both were present within the same cell. Swapping of the ori is a common practice; however, it is vital that all regions of the original replicon be removed. Additional vector pSAMRNAI illustrated that incompatibility remains when portions of the replicon, such as RNAI and/or Rom, are retained; pSAMRNAI, which contains the intact RNAI but not ROM, lowered the copy number of pGEX-6p-1 to 18±2 in doubly transformed cells due to retention of the pET-28a(+)-derived RNAI. Thus, pSAMRNAI is incompatible with vectors controlled by the pBR322 replicon and further demonstrates the need to remove all portions of the original replicon and to quantitatively assess copy number, both individually and in combination, to ensure vector compatibility. To our knowledge, this is the first instance where the nascent vector has been quantitatively assessed for both plasmid copy number and compatibility. New vector pSAM provides ease of transferring sequences from commonly used pET-28a(+) into a vector compatible with the pBR322 family of plasmids. This essential need is currently not filled.

The bZIP dimer localizes at DNA full-sites where each basic region can alternately translocate and bind to subsites at the half-site.

Crystal structures of the GCN4 bZIP (basic region/leucine zipper) with the AP-1 or CRE site show how each GCN4 basic region binds to a 4 bp cognate half-site as a single DNA target; however, this may not always fully describe how bZIP proteins interact with their target sites. Previously, we showed that the GCN4 basic region interacts with all 5 bp in half-site TTGCG (termed 5H-LR) and that 5H-LR comprises two 4 bp subsites, TTGC and TGCG, which individually are also target sites of the basic region. In this work, we explore how the basic region interacts with 5H-LR when the bZIP dimer localizes to full-sites. Using AMBER molecular modeling, we simulated GCN4 bZIP complexes with full-sites containing 5H-LR to investigate in silico the interface between the basic region and 5H-LR. We also performed in vitro investigation of bZIP-DNA interactions at a number of full-sites that contain 5H-LR versus either subsite: we analyzed results from DNase I footprinting and electrophoretic mobility shift assay (EMSA) and from EMSA titrations to quantify binding affinities. Our computational and experimental results together support a highly dynamic DNA-binding model: when a bZIP dimer localizes to its target full-site, the basic region can alternately recognize either subsite as a distinct target at 5H-LR and translocate between the subsites, potentially by sliding and hopping. This model provides added insights into how α-helical DNA-binding domains of transcription factors can localize to their gene regulatory sequences in vivo.

Crystal structure of the minimalist Max-E47 protein chimera.

Max-E47 is a protein chimera generated from the fusion of the DNA-binding basic region of Max and the dimerization region of E47, both members of the basic region/helix-loop-helix (bHLH) superfamily of transcription factors. Like native Max, Max-E47 binds with high affinity and specificity to the E-box site, 5'-CACGTG, both in vivo and in vitro. We have determined the crystal structure of Max-E47 at 1.7 Å resolution, and found that it associates to form a well-structured dimer even in the absence of its cognate DNA. Analytical ultracentrifugation confirms that Max-E47 is dimeric even at low micromolar concentrations, indicating that the Max-E47 dimer is stable in the absence of DNA. Circular dichroism analysis demonstrates that both non-specific DNA and the E-box site induce similar levels of helical secondary structure in Max-E47. These results suggest that Max-E47 may bind to the E-box following the two-step mechanism proposed for other bHLH proteins. In this mechanism, a rapid step where protein binds to DNA without sequence specificity is followed by a slow step where specific protein:DNA interactions are fine-tuned, leading to sequence-specific recognition. Collectively, these results show that the designed Max-E47 protein chimera behaves both structurally and functionally like its native counterparts.

Forced homodimerization of the c-Fos leucine zipper in designed bHLHZ-like hybrid proteins MaxbHLH-Fos and ArntbHLH-Fos.

Although the c-Fos leucine zipper (LZ) does not form a homodimer in its native basic region/leucine zipper (bZIP) structure, we found that it is capable of homodimerization and promoting protein folding in engineered basic region/helix-loop-helix/leucine zipper (bHLHZ) hybrid proteins MaxbHLH-Fos and ArntbHLH-Fos, in which the bHLH subdomains of Max and Arnt are fused to the c-Fos LZ. By using the in vivo yeast one-hybrid system and in vitro circular dichroism and quantitative fluorescence anisotropy, we demonstrated that attachment of the c-Fos LZ to the otherwise unstructured MaxbHLH resulted in a hybrid bHLHZ-like protein now competent for homodimerization and DNA binding at the cognate E-box site, CACGTG. In ArntbHLH-Fos, the c-Fos LZ promoted proper folding of the HLH structure, although unlike MaxbHLH, ArntbHLH alone is capable of homodimerization and DNA binding. In addition, by comparing the E-box binding and secondary structures of MaxbHLH-Fos and two derivatives containing targeted mutations in the c-Fos LZ, we found that cooperative communication exists between the bHLH and LZ: proper folding of the four-helix bundle in the HLH region could be induced by the c-Fos LZ, and the HLH dimerization region could force homodimerization of the c-Fos LZ. These results demonstrate that although intrinsically unfavorable, the c-Fos LZ can homodimerize, demonstrating that the same c-Fos LZ element can yield orthogonal differences in structure and/or DNA-binding function within different transcription factor families, including the bZIP and bHLHZ.

FRep: a fluorescent protein-based bioprobe for in vivo detection of protein-DNA interactions.

We describe a bacterial reporter system, FRep, for rapid and facile detection of protein-DNA recognition. The bioprobe reporter comprises genes of two fluorescent proteins (FPs) separated by a potential DNA target. If a coexpressed transcription factor binds the DNA target, transcription of the second FP is impeded, resulting in loss of FRET partner. Using ratiometric FRET, we show that evaluation of protein-DNA recognition can be reliably made on bZIP and bHLHZ transcription factors and their DNA targets. FRep displays similar thresholds of detection regarding protein-DNA binding affinities, as compared to well-established electrophoretic and yeast assays, although we observed variations in the intensity of fluorescence signals and detection thresholds that may depend on differences between DNA-binding protein production levels and/or stability in the cell, or the expressed bioprobe linker between the two FPs. FRep can potentially be applied to high-throughput searches of both protein and DNA libraries; in a mock library screen, binding and nonbinding complexes can even be distinguished by visual inspection of colonies on plates. FRep presents notable advantages over existing technologies when applied to assessing protein-DNA interactions in vivo, and this approach has the potential for applications in assaying protein-protein interactions and screening molecules that influence specific macromolecular interactions.

Reengineering natural design by rational design and in vivo library selection: the HLH subdomain in bHLHZ proteins is a unique requirement for DNA-binding function.

To explore the role of the HLH subdomain in bHLHZ proteins, we designed sets of minimalist proteins based on bHLHZ protein Max, bHLH/PAS protein Arnt and bZIP protein C/EBP. In the first, the Max bHLH and C/EBP leucine zipper were fused such that the leucine heptad repeats were not in register; therefore, the protein dimerization interface was disrupted. Max1bHLH-C/EBP showed little ability to activate transcription from the E-box (5'-CACGTG) in the yeast one-hybrid assay, and no E-box binding by quantitative fluorescence anisotropy. Max1bHLH-C/EBP's activity was significantly improved after library selection (three amino acids randomized between HLH and leucine zipper), despite the Max bHLH and C/EBP zipper still being out of register: a representative mutant gave a high nanomolar K(d) value for E-box binding. Thus, selection proved to be a powerful tool for salvaging the flawed Max1bHLH-C/EBP, although the out-of-register mutants still did not achieve the strong DNA-binding affinities displayed by their in-register counterparts. ArntbHLH-C/EBP hybrids further demonstrated the importance of maintaining register, as out-of-register mutants showed no E-box-responsive activity, whereas the in-register hybrid showed moderate activity. In another design, we eliminated the HLH altogether and fused the Max basic region to the C/EBP zipper to generate bZIP-like hybrids. Despite numerous designs and selections, these hybrids possessed no E-box-responsive activity. Finally, we tested the importance of the loop sequence in MaxbHLHZ by fluorescence and circular dichroism. In one mutant, the loop was shortened by two residues; in the other, the Lys57:DNA-backbone interaction was abolished by mutation to Gly57. Both showed markedly decreased E-box-binding relative to MaxbHLHZ. Our results suggest that, in contrast to the more rigid bZIP, the HLH is capable of significant conformational adaptation to enable gene-regulatory function and is required for protein dimerization and positioning the basic region for DNA recognition.

Max-E47, a designed minimalist protein that targets the E-box DNA site in vivo and in vitro.

Max-E47 is a designed hybrid protein comprising the Max DNA-binding basic region and E47 HLH dimerization subdomain. In the yeast one-hybrid system (Y1H), Max-E47 shows strong transcriptional activation from the E-box site, 5'-CACGTG, targeted by the Myc/Max/Mad network of transcription factors; two mutants, Max-E47Y and Max-E47YF, activate more weakly from the E-box in the Y1H. Quantitative fluorescence anisotropy titrations to gain free energies of protein:DNA binding gave low nanomolar K(d) values for the native MaxbHLHZ, Max-E47, and the Y and YF mutants binding to the E-box site (14, 15, 9, and 6 nM, respectively), with no detectable binding to a nonspecific control duplex. Because these minimalist, E-box-binding hybrids have no activation domain and no interactions with the c-MycbHLHZ, as shown by the yeast two-hybrid assay, they can potentially serve as dominant-negative inhibitors that suppress activation of E-box-responsive genes targeted by transcription factors including the c-Myc/Max complex. As proof-of-principle, we used our modified Y1H, which allows direct competition between two proteins vying for a DNA target, to show that Max-E47 effectively outcompetes the native MaxbHLHZ for the E-box; weaker competition is observed from the two mutants, consistent with Y1H results. These hybrids provide a minimalist scaffold for further exploration of the relationship between protein structure and DNA-binding function and may have applications as protein therapeutics or biochemical probes capable of targeting the E-box site.

Hybrids of the bHLH and bZIP protein motifs display different DNA-binding activities in vivo vs. in vitro.

Minimalist hybrids comprising the DNA-binding domain of bHLH/PAS (basic-helix-loop-helix/Per-Arnt-Sim) protein Arnt fused to the leucine zipper (LZ) dimerization domain from bZIP (basic region-leucine zipper) protein C/EBP were designed to bind the E-box DNA site, CACGTG, targeted by bHLHZ (basic-helix-loop-helix-zipper) proteins Myc and Max, as well as the Arnt homodimer. The bHLHZ-like structure of ArntbHLH-C/EBP comprises the Arnt bHLH domain fused to the C/EBP LZ: i.e. swap of the 330 aa PAS domain for the 29 aa LZ. In the yeast one-hybrid assay (Y1H), transcriptional activation from the E-box was strong by ArntbHLH-C/EBP, and undetectable for the truncated ArntbHLH (PAS removed), as detected via readout from the HIS3 and lacZ reporters. In contrast, fluorescence anisotropy titrations showed affinities for the E-box with ArntbHLH-C/EBP and ArntbHLH comparable to other transcription factors (K(d) 148.9 nM and 40.2 nM, respectively), but only under select conditions that maintained folded protein. Although in vivo yeast results and in vitro spectroscopic studies for ArntbHLH-C/EBP targeting the E-box correlate well, the same does not hold for ArntbHLH. As circular dichroism confirms that ArntbHLH-C/EBP is a much more strongly alpha-helical structure than ArntbHLH, we conclude that the nonfunctional ArntbHLH in the Y1H must be due to misfolding, leading to the false negative that this protein is incapable of targeting the E-box. Many experiments, including protein design and selections from large libraries, depend on protein domains remaining well-behaved in the nonnative experimental environment, especially small motifs like the bHLH (60-70 aa). Interestingly, a short helical LZ can serve as a folding- and/or solubility-enhancing tag, an important device given the focus of current research on exploration of vast networks of biomolecular interactions.

The bZIP targets overlapping DNA subsites within a half-site, resulting in increased binding affinities.

We previously reported that the wt bZIP, a hybrid of the GCN4 basic region and C/EBP leucine zipper, not only recognizes GCN4 cognate site AP-1 (TGACTCA) but also selectively targets noncognate DNA sites, in particular the C/EBP site (TTGCGCAA). In this work, we used electrophoretic mobility shift assay and DNase I footprinting to investigate the factors driving the high affinity between the wt bZIP and the C/EBP site. We found that on each strand of the C/EBP site, the wt bZIP recognizes two 4 bp subsites, TTGC and TGCG, which overlap to form the effective 5 bp half-site (TTGCG). The affinity of the wt bZIP for the overall 5 bp half-site is >or=10-fold stronger than that for either 4 bp subsite. Our results suggest that interactions of the wt bZIP with both subsites contribute to the strong affinity at the overall 5 bp half-site and, consequently, the C/EBP site. Accordingly, we propose that the wt bZIP undergoes conformational changes to slide between the two overlapping subsites on the same DNA strand and establish sequence-selective contacts with the different subsites. The proposed binding mechanism expands our understanding of what constitutes an actual DNA target site in protein-DNA interactions.

AhR/Arnt:XRE interaction: turning false negatives into true positives in the modified yeast one-hybrid assay.

Given the frequent occurrence of false negatives in yeast genetic assays, it is both interesting and practical to address the possible mechanisms of false negatives and, more important, to turn false negatives into true positives. We recently developed a modified yeast one-hybrid system (MY1H) useful for investigation of simultaneous protein-protein and protein:DNA interactions in vivo. We coexpressed the basic helix-loop-helix/Per-Arnt-Sim (bHLH/PAS) domains of aryl hydrocarbon receptor (AhR) and aryl hydrocarbon receptor nuclear translocator (Arnt)--namely NAhR and NArnt, respectively--which are known to form heterodimers and bind the cognate xenobiotic response element (XRE) sequence both in vitro and in vivo, as a positive control in the study of XRE-binding proteins in the MY1H system. However, we observed negative results, that is, no positive signal detected from binding of the NAhR/NArnt heterodimer and XRE site. We demonstrate that by increasing the copy number of XRE sites integrated into the yeast genome and using double GAL4 activation domains, the NAhR/NArnt heterodimer forms and specifically binds the cognate XRE sequence, an interaction that is now clearly detectable in the MY1H system. This methodology may be helpful in troubleshooting and correcting false negatives that arise from unproductive transcription in yeast genetic assays.

Enhancing the specificity of the enterokinase cleavage reaction to promote efficient cleavage of a fusion tag.

In our work with designed minimalist proteins based on the bZIP motif, we have found our His-tagged proteins to be prone to inclusion body formation and aggregation; we suspect this problem is largely due to the His tag, known to promote aggregation. Using AhR6-C/EBP, a hybrid of the AhR basic region and C/EBP leucine zipper, as representative of our bZIP-like protein family, we attempted removal of the His tag with enterokinase (EK) but obtained the desired cleavage product in very small yield. EK is known for proteolysis at noncanonical sites, and most cleavage occurred at unintended sites. We manipulated experimental conditions to improve specificity of proteolysis and analyzed the cleavage products; no effect was observed after changing pH, temperature, or the amount of EK. We then suspected the accessibility of the EK site was impeded due to protein aggregation. We found that the easily implemented strategy of addition of urea (1-4 M) greatly improved EK cleavage specificity at the canonical site and reduced adventitious cleavage. We believe that this enhancement in specificity is due to a more "open" protein structure, in which the now accessible canonical target can compete effectively with adventitious cleavage sites of related sequence.

The GCN4 bZIP targets noncognate gene regulatory sequences: quantitative investigation of binding at full and half sites.

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 Kd values of 13 and 12 nM, respectively, to noncognate sites with Kd values of 120 nM to low microM. DNA-binding selectivity at half sites is maintained; however, half-site binding affinities sharply decrease from the cognate half site (Kd = 84 nM) to noncognate half sites (all Kd values > 2 microM). CD shows that comparable levels of alpha-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, alpha-helical proteins with desired DNA recognition properties.