Design of temperature-sensitive penicillinase repressors by replacement of Pro in predicted beta-turn structures.
Pro residues in predicted beta-turn structures were substituted with other amino acids to obtain temperature-sensitive penicillinase repressors (PenI). A mutant repressor (P70L; Pro-70 is substituted with Leu) was inactive at 48 degrees C and penP gene expression was derepressed (1,200 U/OD660 [optical density at 660 nm] ), although the mutant was still active at 30 degrees C (27 U). The heat induction ratio (penicillinase activity at 48 degrees C compared with that at 30 degrees C) of the mutant was 98 times higher than that of the wild type (i.e., 44 versus 0.45). This result indicated that the side chain of the Leu residue in P70L destroyed the proper folding of the repressor protein at the elevated temperature, whereas the Pro residue of the wild-type repressor stabilized this predicted beta-turn structure even at 48 degrees C. When the Pro residue was replaced by amino acid residues with smaller side chains (i.e., Gly and Ala), these mutant repressors were less temperature sensitive than P70L. These data suggest that the presence of the Pro residue in the beta-turn structure could be one of the key factors in stabilizing protein structure at elevated temperatures.
Imanaka
T
T
Department of Biotechnology, Faculty of Engineering, Osaka University, Japan.
Nakae
M
M
Ohta
T
T
Takagi
M
M
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D015152:Blotting, Northern; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010405:Penicillinase / Q000235:genetics*; D011392:Proline / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D013696:Temperature
D012097:Repressor Proteins; D011392:Proline; D010405:Penicillinase