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Nucleic Acids Research 2005 33(18):5896-5903; doi:10.1093/nar/gki899
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Published online 13 October 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oxfordjournals.org

Article

Switching DNA-binding specificity by unnatural amino acid substitution

Atanu Maiti and Siddhartha Roy1,*

Department of Biophysics, Bose Institute P-1/12, C.I.T. Scheme VII M, Kolkata 700 054, India 1Indian Institute of Chemical Biology 4, Raja Subodh Mullick Road, Kolkata 700 032, India

*To whom correspondence should be addressed. Tel: +91 33 2413 1157; Fax: +91 33 2473 5197; Email: siddhartharoy{at}iicb.res.in

Received July 21, 2005. Revised September 17, 2005. Accepted September 28, 2005.

The specificity of protein–nucleic acid recognition is believed to originate largely from hydrogen bonding between protein polar atoms, primarily side-chain and polar atoms of nucleic acid bases. One way to design new nucleic acid binding proteins of novel specificity is by structure-guided alterations of the hydrogen bonding patterns of a nucleic acid–protein complex. We have used cI repressor of bacteriophage {lambda} as a model system. In the {lambda}-repressor–DNA complex, the {varepsilon}-NH2 group (hydrogen bond donor) of lysine-4 of {lambda}-repressor forms hydrogen bonds with the amide carbonyl atom of asparagine-55 (acceptor) and the O6 (acceptor) of CG6 of operator site OL1. Substitution of lysine-4 (two donors) by iso-steric S-(2-hydroxyethyl)-cysteine (one donor and one acceptor), by site-directed mutagenesis and chemical modification, leads to switch of binding specificity of {lambda}-repressor from C:G to T:A at position 6 of OL1. This suggests that unnatural amino acid substitutions could be a simple way of generating nucleic acid binding proteins of altered specificity.


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