caa a22 4500 37892169X CHVBK 20180305123607.0 cr unu---uuuuu 161128e20040101xx s 000 0 eng 10.1351/pac200476071579 doi (NATIONALLICENCE)gruyter-10.1351/pac200476071579 Shin J. A. Department of Chemistry, University of Toronto, Mississauga, Ontario L5L 1C6, Canada Minimalist proteins: Design of new molecular recognition scaffolds [Elektronische Daten] [J. A. Shin] 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-bind- ing 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. © 2013 Walter de Gruyter GmbH, Berlin/Boston Pure and Applied Chemistry De Gruyter 76/7-8(2004-01-01), 1579-1590 0033-4545 76:7-8<1579 2004 76 pac https://doi.org/10.1351/pac200476071579 text/html Onlinezugriff via DOI 1 research article jats NATIONALLICENCE 856 40 https://doi.org/10.1351/pac200476071579 text/html Onlinezugriff via DOI NATIONALLICENCE 100 1- Shin J. A. Department of Chemistry, University of Toronto, Mississauga, Ontario L5L 1C6, Canada NATIONALLICENCE 773 0- Pure and Applied Chemistry De Gruyter 76/7-8(2004-01-01), 1579-1590 0033-4545 76:7-8<1579 2004 76 pac CC0 http://creativecommons.org/publicdomain/zero/1.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-gruyter