The 28.3 kDa FK506 binding protein from a thermophilic archaeum, Methanobacterium thermoautotrophicum, protects the denaturation of proteins in vitro

Abstract

Two families of FK506 binding protein (FKBP) type peptidyl-prolyl cis±trans isomerase (PPIase) have been found in Archaea. One is the 16±18 kDa short type FKBP family, and another is the 26±30 kDa long type FKBP family. The latter has a longer C-terminal region than the former. In this study, the 28.3 kDa long type FKBP gene from a thermophilic archaeum, Methanobacterium thermoautotrophicum, was expressed in Escherichia coli, and its gene product (MbFK) was characterized. The PPIase activity of MbFK was much lower than those of other FKBPs reported against oligopeptidyl substrates. MbFK protected green fluorescent protein (GFP) and rhodanese from thermal denaturation. Furthermore, MbFK suppressed the aggregation of chemically unfolded rhodanese and elevated the yield of its refolding although this activity was weaker than that of GroEL / ES. We made two deletion mutants, MbFK-N which lacked the C-terminal region, and MbFK-C which had only the C-terminal region. Far-UV CD spectra of these mutants showed that their secondary structures did not change from that of the wild-type. Whereas the PPIase activity of MbFK-N was low but detectable, that of MbFK-C was undetectable. The MbFK-C protected the thermal protein aggregation, and possessed a weak but significant aggregation suppressing activity against chemically unfolded protein. However, the MbFK-N did not suppress the aggregation of chemically unfolded rhodanese while it protected heat induced aggregation of rhodanese. These results may indicate that aggregation suppressing activity of MbFK-W against chemically unfolded protein are exerted mainly by its C-terminal domain while both domains contribute to thermal protein aggregation suppression.

Structure-Function Analysis of PrsA Reveals Roles for the Parvulin-like and Flanking N- and C-terminal Domains in Protein Folding and Secretion in Bacillus subtilis

“…Refolding of Chemically Unfolded Rhodanese-Unfolding and refolding of rhodanese was carried out essentially as previously described (32). Rhodanese (Sigma Aldrich) was incubated at 38 M concentration in a buffer containing 50 mM Tris-HCl, pH 7.8, 6 M GdnHCl, and 5 mM dithiothreitol for 1 h at 25°C.…”

FK506 Binding Protein from the Hyperthermophilic Archaeon Pyrococcus horikoshii Suppresses the Aggregation of Proteins in Escherichia coli

“…N-Succinyl-Ala-Leu-Pro-Phe-p-nitroanilide (N-suc-ALPF) was purchased from Peptide Institute, Inc. (Osaka, Japan). MbtFKBP28 was prepared as described previously (12). The rabbit antiserum against FKBP from P. horikoshii (PhFKBP29) was prepared by Takara Shuzo Co. (Kyoto, Japan).…”

“…Cells of P. horikoshii were harvested by centrifugation from 300 l of a cell suspension obtained from the Japan Collection of Microorganisms (Riken, Saitama, Japan). The genomic DNA was prepared from the cells according to a previously described procedure (12) and used for the PCR template. The gene for FKBP from P. horikoshii (PhFKBP29) was amplified by PCR with the primer set of PhFK-F1 and PhFK-R1 (Table 1).…”

“…After we confirmed the stability of the FKBP to chymotrypsin digestion, we measured the PPIase activity by protease-coupled assay with the oligopeptides N-suc-ALPF and N-suc-AAPF as substrates (3,23). The PPIase assay of 5 to 80 M PhFKBP29 was carried out as described previously (12).…”

“…100 amino acids (20). A long-type FKBP from Methanobacterium thermoautotrophicum (Mbt-FKBP28, gene number MTH1125 [http://www.ncbi.nlm.nih .gov/]) has shown detectable but very low PPIase activity, in contrast to the short-type FKBPs, while it showed weak but significant chaperone-like protein folding activity with protein aggregation suppression in vitro (12). A genome sequence analysis has indicated that the hyperthermophilic archaea Pyrococcus horikoshii, Archaeoglobus fulgidus, and Aeropyrum pernix have only the long-type FKBP gene as a PPIase in their genomes (20).…”