Foldability, enzymatic activity, and interacting ability of barnase mutants obtained by permutation of secondary structure units.
Barnase, a well-characterized ribonuclease, has been decomposed into six modules (M1-M6) or secondary structure units (S1-S6). We have studied the foldability and activity of the barnase mutants obtained by permutation of the four internal modules (M2-M5) or secondary structure units (S2-S5) to investigate whether permutation of these building blocks is a useful way to create foldable and/or functional proteins. In this study, we found that one of the secondary structure unit mutants was expressed in Escherichia coli only when His102 was substituted by alanine, which is a catalytic residue of wild-type barnase. This mutant (S2354H102A) had ordered conformations, which unfolded cooperatively during urea-induced unfolding experiments. S2354H102A interacted with other barnase mutants to show a distinct RNase activity, although its own activity was quite weak. This interaction was specific, because S2354H102A interacted with only barnase mutants having His 102 and certain orders of the secondary structure units giving a distinct RNase activity. These results suggest that secondary structure units permuted in barnase mutants maintain their intrinsic "interacting ability" that is used for the folding of wild-type barnase, and the units can form certain conformations that complement those of the appropriate counterparts. Seven of 23 secondary structure unit mutants and only 2 of 23 module mutants had RNase activity. On the basis of the results of analyses of foldability and RNase activity of the mutants performed in this and previous studies, we conclude that secondary structure units are more suitable than modules as building blocks to create novel foldable and/or functional proteins in the case of barnase.
Tsuji
Toru
T
Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University, 3-14-1, Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan.
Yanagawa
Hiroshi
H
D000409:Alanine / Q000737:chemistry / Q000235:genetics; D019943:Amino Acid Substitution; D001426:Bacterial Proteins; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006639:Histidine / Q000737:chemistry / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics*; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012260:Ribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014508:Urea
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D006639:Histidine; D014508:Urea; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease; D000409:Alanine