Engineering of monomeric FK506‐binding protein 22 with peptidyl prolyl cis‐trans isomerase

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Abstract
FK506‐binding protein 22 (FKBP22) from the psychrotrophic bacterium Shewanella sp. SIB1 is a homodimeric protein with peptidyl prolyl cis–trans isomerase (PPIase) (EC 5.2.1.8) activity. Each monomer consists of 205 amino acid residues. According to a tertiary model, SIB1 FKBP22 assumes a V‐shaped structure, in which two monomers interact with each other at their N‐termini. Each monomer consists of an N‐terminal domain with a dimerization core and a C‐terminal catalytic domain, which are separated by a 40‐residue‐long α‐helix. To clarify the role of this V‐shaped structure, we constructed a mutant protein, in which the N‐domain is tandemly repeated through a flexible linker. This protein, termed NNC‐FKBP22, is designed such that two repetitive N‐domains are folded into a structure similar to that of the Shewanella sp. SIB1 FKBP22 wild‐type protein (WT). NNC‐FKBP22 was overproduced in Escherichia coli in a His‐tagged form, purified and biochemically characterized. Gel‐filtration chromatography and ultracentrifugation analyses indicate that NNC‐FKBP22 exists as a monomer. Analysis of thermal denaturation using differential scanning calorimetry indicates that NNC‐FKBP22 unfolds with two transitions, as does the WT protein. NNC‐FKBP22 exhibited PPIase activity for both peptide and protein substrates. However, in contrast to its activity for peptide substrate, which was comparable to that of the WT protein, its activity for protein substrate was reduced by five‐ to six‐fold, compared to that of the WT. Surface plasmon resonance analyses indicate that NNC‐FKBP22 binds to a reduced form of α‐lactalbumin with a six‐fold weaker affinity than that of WT. These results suggest that a V‐shaped structure of SIB1 FKBP22 is important for efficient binding to a protein substrate. Structured digital abstract • MINT‐7136140: FKBP22 (uniprotkb:Q765B0) binds (MI:0407) to Alpha‐lactalbumin (uniprotkb:P00711) by surface plasmon resonance (MI:0107)

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