Stabilities and activities of the N- and C-domains of FKBP22 from a psychrotrophic bacterium overproduced in Escherichia coli.
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 alpha3 helix spans these domains. An N-domain with the entire alpha3 helix (N-domain+) and a C-domain with the entire alpha3 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 degrees 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 32.5 degrees C and 46.6 degrees C for the intact protein, and single heat absorption peaks at 44.7 degrees C for N-domain+ and 35.6 degrees C for C-domain+. These results indicate that the thermal unfolding transitions of the intact protein at lower and higher temperatures represent those of C- and N-domains, respectively. Because the unfolding temperature of C-domain+ is much higher than its optimum temperature for activity, SIB1 FKBP22 may adapt to low temperatures by increasing a local flexibility around the active site. This study revealed the relationship between the stability and the activity of a psychrotrophic FKBP22.
Suzuki
Yutaka
Y
Department of Material and Life Science, Graduate School of Engineering, Osaka University, Suita, Osaka, Japan.
Takano
Kazufumi
K
Kanaya
Shigenori
S
D001483:Base Sequence; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D008958:Models, Molecular; D008970:Molecular Weight; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020592:Shewanella / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D022021:Tacrolimus Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D017931:DNA Primers; D011994:Recombinant Proteins; C403422:FKBP22; D022021:Tacrolimus Binding Proteins