Sequence-specific recognition of DNA by hydrophobic, alanine-rich mutants of the basic region/leucine zipper motif investigated by fluorescence anisotropy

We generated minimalist proteins capable of sequence‐specific, high‐affinity binding of DNA to probe how proteins are used and can be used to recognize DNA. In order to quantify binding affinities and specificities in our protein–DNA system, we used fluorescence anisotropy to measure in situ the thermodynamics of binding of alanine‐rich mutants of the GCN4 basic region/leucine zipper (bZIP) domain to DNA duplexes containing target sites AP‐1 (5′‐TGACTCA‐3′) or ATF/CREB (5′‐TGACGTCA‐3′). We simplified the α‐helical bZIP molecular recognition scaffold by alanine substitution: 4A, 11A, and 18A contain four, eleven, and eighteen alanine mutations in their DNA‐binding basic regions, respectively. DNase I footprinting analysis demonstrates that all bZIP mutants retain the sequence‐specific DNA‐binding function of native GCN4 bZIP. Titration of fluorescein‐labeled oligonucleotide duplexes with increasing amounts of protein yielded low nanomolar dissociation constants for all bZIP mutants in complex with the AP‐1 and ATF/CREB sites: binding to the nonspecific control duplex was > 1000‐fold weaker. Remarkably, the most heavily mutated protein 18A, containing 24 alanines in its 27‐residue basic region, still binds AP‐1 and ATF/CREB with dissociation constants of 15 and 7.8 nM, respectively. Similarly, wild‐type bZIP binds these sites with Kd values of 9.1 and 14 nM. 11A also displays low nanomolar dissociation constants for AP‐1 and ATF/CREB, while 4A binds these sites with ∼ 10‐fold weaker Kd values. Thus, both DNA‐binding specificity and affinity are maintained in all our bZIP derivatives. This Ala‐rich scaffold may be useful in design and synthesis of small α‐helical proteins with desired DNA‐recognition properties capable of serving as therapeutics targeting transcription. © 2002 Wiley Periodicals, Inc. Biopolymers 65: 10–20, 2002