Folding mechanism of a protein from a hyperthermophile with unusually slow folding rates

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We have hound that the refolding reaction of pyrrolidone carboxyl peptidase (PCP) from a hyperthermophile, Pyrococcus furiosus, is unusually slow at acidic pH and can be controlled by regulating the incubation temperature ; the refolding reaction significantly stops at pH 2.3 and 4 ℃. In this period, in order to elucidate the folding mechanism of a protein from a hyperthermophile with unusually slow folding rates, we have completed two papers using cysteine-free PCP (PCP-OSH) with unusually slow refolding rates. PCP-OSH was used in place of PCP to avoid troubles due to the formation of disulfide bonds. (1) PCPs from hyperthermophiles have a structurally conserved and completely buried Glu192 in the hydrophobic core ; in contrast, the corresponding residue in mesophile protein is a hydrophobic residue, He. To elucidate the role of the buried Glu in stability and folding rates of PCP from hyperthermophiles, we examined changes in stability and structure due to mutations at Glu192. The results indicated that completely buried Glu192 contributes to stabilization of PCP-OSH due to the formation of strong intramolecular hydrogen bonds, and the hydrogen bonds by the non-ionized and buried Glu can contribute more than the burial of hydrophobic groups to the conformational stability of proteins (Kausahik et al., 2006). (2) The above denatured state (D_1 state) of PCP corresponds to the denatured structure that exists in equilibrium with the native state under physiological conditions. To elucidate the structural basis of the D_1 state, H/D exchange experiments with PCP-OSH were performed at pD 3.4 and 4 ℃. The results indicated that amide protons in the C-terminal a6-helix region hardly exchanged in the D_1 state with deuterium even after 7 days, suggesting that the a6-helix (from Ser188 to Glu205) of PCP-OSH was stably formed in the D_1 state (Iimura et al., 2007).