title: Circularly permuted dihydrofolate reductase of E. coli has functional activity and a destabilized tertiary structure.
abstract: Three circularly permuted variants of Escherichia coli dihydrofolate reductase genes were constructed. Linkers coding tri- and pentapeptides were used to connect the natural 5'- and 3'-terminal ends. Only one variant of circularly permuted protein with tripeptide linker and the cleavage of the peptide bond between 107 and 108 amino acid residues was produced in a good yield. The expressed protein was insoluble in the cells, but at pH 8.0 and higher the isolated protein was soluble. Enzymatic assay and physical studies have shown that permuted dihydrofolate reductase has a destabilized tertiary structure. Only the addition of the natural substrates or inhibitors lead to the protein with the native-like structure and functional activity.
authors.affiliation: Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region.
authors.lastname: Protasova NYu
authors.forename: M L
authors.initials: ML
authors.lastname: Kireeva
authors.forename: N V
authors.initials: NV
authors.lastname: Murzina
authors.forename: A G
authors.initials: AG
authors.lastname: Murzin
authors.forename: V N
authors.initials: VN
authors.lastname: Uversky
authors.forename: O I
authors.initials: OI
authors.lastname: Gryaznova
authors.forename: A T
authors.initials: AT
authors.lastname: Gudkov
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002942:Circular Dichroism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics; D014508:Urea / Q000494:pharmacology
chemical_list: D011994:Recombinant Proteins; D014508:Urea; D013762:Tetrahydrofolate Dehydrogenase