N- and C-terminal fragments of a globular protein constructed by elongation of modules as a units associated for functional complementation.
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.
Tsuji
Toru
T
Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University, Kohoku-ku, Yokohama, Japan.
Nagata
Takashi
T
Yanagawa
Hiroshi
H
D001426:Bacterial Proteins; D002942:Circular Dichroism; D019020:Directed Molecular Evolution; D005091:Exons; D005816:Genetic Complementation Test; D007700:Kinetics; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D019151:Peptide Library; D011489:Protein Denaturation; D012260:Ribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D010446:Peptide Fragments; D019151:Peptide Library; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease