Minimalist proteins: Design of new molecular recognition scaffolds
J. A. Shin
Department of Chemistry, University of Toronto, Mississauga,
Ontario L5L 1C6, Canada
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-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.
*Lecture presented at the symposium "Chemistry of nucleic acids", as part of the 39th IUPAC Congress and 86th Conference of the Canadian Society for Chemistry: Chemistry at the Interfaces, Ottawa, Canada, 10-15 August 2003. Other Congress presentations are published in this issue, pp. 1295-1603.
Page last modified 15 September 2004.
Copyright © 2004 International Union of Pure and Applied Chemistry.
Questions or comments about IUPAC, please contact, the Secretariat.
Questions regarding the website, please contact web
manager.