Abstract
We hypothesize that we can exploit what Nature has already evolved by manipulating the alpha-helix molecular recognition 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. 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. The proteins generated, wt bZIP, 4A, 11A, and 18A, contain 0, 4, 11, and 18 alanine mutations in their DNA-binding basic regions, respectively. All alanine mutants still retain alpha-helical structure and DNA-binding function, despite loss of virtually all Coulombic protein-DNA interactions. Mass spectrometry allowed characterization of proteins and post-translational modifications. 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. The roles of van der Waals and Coulombic interactions toward binding specificity and affinity are being investigated. 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, alpha-helical proteins with desired DNA-recognition properties.