tailieunhanh - Báo cáo khoa học: Stabilities and activities of the N- and C-domains of FKBP22 from a psychrotrophic bacterium overproduced in Escherichia coli

FKBP22 from a psychrotrophic bacteriumShewanellasp. SIB1, is a dimer-ic protein with peptidyl prolyl cis-transisomerase (PPIase) activity. Accord-ing to homology modeling, it consists of an N-terminal domain, which is involved in dimerization of the protein, and a C-terminal catalytic domain. A longa3 helix spans these domains. An N-domain with the entirea3 helix (N-domain + ) and a C-domain with the entire a3 helix (C-domain + ) were overproduced inEscherichia coliin a His-tagged form, purified, and their biochemical properties were compared with those of the intact protein. C-domain + was shown to be a monomer and enzymatically active. Its opti-mum temperature for activity (10 C) was identical to that of the intact protein. . | iFEBS Journal Stabilities and activities of the N- and C-domains of FKBP22 from a psychrotrophic bacterium overproduced in Escherichia coli Yutaka Suzuki1 Kazufumi Takano1 2 and Shigenori Kanaya1 1 Department of Materialand Life Science Graduate Schoolof Engineering Osaka University Suita Osaka Japan 2 PRESTO JST Suita Osaka Japan Keywords domain structure FKBP22 PPIase psychrotrophic bacterium thermal stability Correspondence S. Kanaya Department of Materialand Life Science Graduate School of Engineering Osaka University 2-1 Yamadaoka Suita Osaka 565-0871 Japan Tel Fax 81 6 6879 7938 E-mail kanaya@ Received 27 September 2004 revised 23 October 2004 accepted 29 October 2004 doi FKBP22 from a psychrotrophic bacterium Shewanella sp. SIB1 is a dimeric protein with peptidyl prolyl cis-trans isomerase PPIase activity. According to homology modeling it consists of an N-terminal domain which is involved in dimerization of the protein and a C-terminal catalytic domain. A long a3 helix spans these domains. An N-domain with the entire a3 helix N-domain and a C-domain with the entire a3 helix C-domain were overproduced in Escherichia coli in a His-tagged form purified and their biochemical properties were compared with those of the intact protein. C-domain was shown to be a monomer and enzymatically active. Its optimum temperature for activity 10 C was identical to that of the intact protein. Determination of the PPIase activity using peptide and protein substrates suggests that dimerization is required to make the protein fully active for the protein substrate or that the N-domain is involved in substrate-binding. The differential scanning calorimetry studies revealed two distinct heat absorption peaks at C and C for the intact protein and single heat absorption peaks at C for N-domain and C for C-domain . These results indicate that the thermal unfolding transitions of the intact protein at lower and