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Journal of Biochemistry Advance Access published online on November 4, 2009
Journal of Biochemistry, doi:10.1093/jb/mvp179
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© The authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Mimicking the evolution of a thermally stable monomeric four-helix bundle by fusion of four identical single-helix peptides

Satoshi Akanuma, Taku Matsuba, Emi Ueno, Naoki Umeda and Akihiko Yamagishi*

Department of Molecular Biology, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.

* Corresponding author: Akihiko Yamagishi, Department of Molecular Biology, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan. Tel: +81-426-76-7139. Fax: +81-426-76-7145. E-mail: yamagish{at}ls.toyaku.ac.jp

Received September 6, 2009; Accepted October 16, 2009


  Abstract

Internal symmetry is a common feature of the tertiary structures of proteins and protein domains. Probably because the genes of homo-oligomeric proteins duplicated and fused, their evolutionary descendants are proteins with internal symmetry. To identify any advantages that cause monomeric proteins with internal symmetry to be selected evolutionarily, we characterized some of the physical properties of a recombinant protein with a sequence consisting of two tandemly fused copies of the Escherichia coli Lac repressor C-terminal {alpha}-helix. This polypeptide exists in solution mainly as dimer that likely maintains a four-helix bundle motif. Thermal unfolding experiments demonstrate that the protein is considerably more stable at elevated temperatures than is a homotetramer consisting of four noncovalently associated copies of a 21-residue polypeptide similar in sequence to that of the Lac repressor C-terminal {alpha}-helix. A tandem duplication of our helix-loop-helix polypeptide yields an even more thermally stable protein. Our results exemplify the concept that fusion of noncovalently assembled polypeptide chains leads to enhanced protein stability. Herein, we discuss how our work relates to the evolutionary selective-advantages realized when symmetrical homo-oligomers evolve into monomers. Moreover, our thermally stable single-chain four-helix bundle protein may provide a robust scaffold for development of new biomaterials.

Key Words: four-helix bundle, fusion, internal symmetry, tandem duplication, thermal stability


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