The leucine zipper dimerizes into the coiled-coil structure shown at the top of the figure the helical zipper then smoothly forks to either side of the DNA major groove. DNA is the horizontal double helix at the bottom of the figure, and the bZIP is the vertical α-helical dimer. (Left) GCN4 bZIP in complex with the AP-1 DNA site, 5′-TGACTCA. In order to begin to probe how Nature uses a pair of α-helices to bind DNA, we ask how DNA can be recognized by the bZIP structure: what is the relationship between a protein’s structure and its DNA-binding function? Can we understand specificity and affinity in protein-DNA complexes by concentrating on simplified natural systems? Can we create a minimalist α-helical protein structure that retains desired DNA-binding function? Nature’s use of the protein α-helix for specific DNA recognition is ubiquitous and maximally utilized by the bZIP, which comprises a pair of short, basic α-helices that recognize the DNA major groove with sequence-specificity and high affinity ( Fig. We exploit the protein α-helix, a structure used ubiquitously for sequence-specific DNA recognition, and one that chemists have successfully used in design and synthesis studies for many years (examples include refs. Our work aims to contribute to understanding the relationship between a protein’s structure and its DNA-binding function-specifically, recognition of the DNA major groove by design of short, simplified α-helices based on the basic region/leucine zipper motif (bZIP). This Ala-rich scaffold may be useful in design and synthesis of small, α-helical proteins with desired DNA-recognition properties. Thus, both DNA-binding specificity and affinity are maintained in all our bZIP derivatives. The roles of van der Waals and Coulombic interactions toward binding specificity and affinity are being investigated. Fluorescence anisotropy and DNase I footprinting were used to measure in situ binding of these mutant proteins to DNA duplexes containing target sites AP-1 (5′-TGACTCA-3′), ATF/CREB (5′-TGACGTCA-3′), or nonspecific DNA. Mass spectrometry allowed characterization of proteins and post-translational modifications. All alanine mutants still retain α-helical structure and DNA-binding function, despite loss of virtually all Coulombic protein-DNA interactions.
The proteins generated, wt bZIP, 4A, 11A, and 18A, contain 0, 4, 11, and 18 alanine mutations in their DNA-binding basic regions, respectively. The already minimal basic region/leucine zipper motif (bZIP) of GCN4 was reduced to an even more simplified structure by substitution with alanine residues-hence, a generic, Ala-based, helical scaffold. Therefore, minimalist proteins capable of sequence-specific, high-affinity binding of DNA were generated to probe how proteins are used and can be used to recognize DNA. We hypothesize that we can exploit what Nature has already evolved by manipulating the α-helix molecular recognition scaffold.
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