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

摘要

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 (1–98), have been constructed by progressive elongation of the modules from the N-terminus. Only M12345 (1–98) had a partially folded conformation, but it lacked detectable RNase activity. A mixture of M12345 (1–98) with M56 (89–110) showed weak but distinct RNase activity. Unfolded M12345 (1–96) was constructed by removal of two residues from the C-terminus of M12345 (1–98). The mixture of M12345 (1–96) with M56 (89–110) also showed RNase activity. Further, the interaction endowed M12345 (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.