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Protein Science (2006), 15:2159-2165. Published by Cold Spring Harbor Laboratory Press. Copyright © 2006 The Protein Society
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Engineered lanthanide-binding metallohomeodomains: Designing folded chimeras by modular turn substitution

Sunghyuk Lim and Sonya J. Franklin

Department of Chemistry, University of Iowa, Iowa City, Iowa 52242, USA

(RECEIVED May 26, 2006; FINAL REVISION June 27, 2006; ACCEPTED June 27, 2006)

A series of chimeric metallohomeodomains are described, engineered by rational design of a flexible Ca/Ln binding site into a DNA-binding scaffold. A modular turn-substitution approach was used to create proteins that both bind DNA and lanthanide ions, while retaining the secondary structure of the full homeodomain (determined by circular dichroism [CD]). Four similar metallohomeodomains were designed (C1–C4), their structural stability predicted by molecular dynamics (MD) simulation of loop-mutations into the known homeodomain structure, and each designed protein cloned, expressed, and purified using standard molecular biology techniques. Two of the four loop insertions resulted in folded, metal- and DNA-binding proteins (EuC2 Kd = 2.1 ± 0.4 µM; EuC4 Kd = 3.2 ± 1.0 µM). These results show the successful incorporation of a metal site into a full protein domain, without compromising long-range structure. This is an important achievement in biomolecular design, as it provides a critical starting point for exploring metallonuclease function and substrate accessibility in a well-organized chimeric protein domain (rather than only in small HTH peptide systems).

Keywords: homeodomain; calmodulin; nuclease; protein engineering; modular design; lanthanides


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