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Journal of Biochemistry Advance Access originally published online on August 7, 2008
Journal of Biochemistry 2008 144(4):513-521; doi:10.1093/jb/mvn099
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© 2008 The Japanese Biochemical Society.

N- and C-terminal Fragments of a Globular Protein Constructed by Elongation of Modules as a Units Associated for Functional Complementation

Toru Tsuji1,*, Takashi Nagata2 and Hiroshi Yanagawa1,{dagger}

1Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University, 3-14-1, Hiyoshi, Kohoku-ku, Yokohama 223-8522; and 2International Graduate School of Arts and Sciences, Yokohama City University, 1-7-29 Suehirocho Tsurumi-ku Yokohama Kanagawa 230-0045, Japan

{dagger}To whom correspondence should be addressed. Tel: +81-45-566-1775, Fax: +81-45-566-1440, E-mail: hyana{at}bio.keio.ac.jp

Received June 4, 2008; Accepted July 17, 2008


  Abstract

We have been interested in partially folded proteins with marginal stability and activity, because they have a potential to be mature proteins by artificial evolution. A module is defined as a contiguous peptide chain forming a compact region in a globular protein. Modules may be used as building blocks to create partially folded proteins. Barnase, a ribonuclease consisting of 110 amino acids, has been divided into six modules (M1–M6), four peptide fragments, M12 (1–52), M123 (1–73), M1234 (1–88) and M12345 [GenBank] (1–98), have been constructed by progressive elongation of the modules from the N-terminus. Only M12345 [GenBank] (1–98) had a partially folded conformation, but it lacked detectable RNase activity. A mixture of M12345 [GenBank] (1–98) with M56 (89–110) showed weak but distinct RNase activity. Unfolded M12345 [GenBank] (1–96) was constructed by removal of two residues from the C-terminus of M12345 [GenBank] (1–98). The mixture of M12345 [GenBank] (1–96) with M56 (89–110) also showed RNase activity. Further, the interaction endowed M12345 [GenBank] (1–96) with conformational stability. We propose that N- and C-terminal fragments obtained by successive elongation of modules would interact to be a complex with marginal stability and activity, which would be used for creating a mature complex by artificial evolution.

Key Words: artificial evolution, exon shuffling, functional complementation, module, protein folding

Abbreviations: ANS, 8-anilino-1-naphthalensulfonic acid; CD, circular dichroism; UV, ultraviolet

*Present address: Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.


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