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Journal of Biochemistry Advance Access published online on August 7, 2008
Journal of Biochemistry, 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

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

Corresponding author: Hiroshi Yanagawa, Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University, 3-14-1, Hiyoshi, Kohoku-ku, Yokohama 223-8522, JAPAN. hyana{at}bio.keio.ac.jp; Tel (81) (45) 566-1775; Fax (81) (45) 566-1440).

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), and 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: exon shuffling, functional complementation, module, artificial evolution, protein folding

Present address: T. Tsuji, Department of Chemistry and Biotechnology, School of engineering, The University of Tokyo. Hongo, Bunkyo-ku, Tokyo 113-8656


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