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The unusually slow relaxation kinetics of the folding-unfolding of pyrrolidone carboxyl peptidase from a hyperthermophile, Pyrococcus furiosus.
J Mol Biol. 2002 Mar 01; 316(4):991-1003.JM

Abstract

In order to understand the thermodynamic and kinetic basis of the intrinsic stability of proteins from hyperthermophiles, the folding-unfolding reactions of cysteine-free pyrrolidone carboxyl peptidase (Cys142/188Ser) (PCP-0SH) from Pyrococcus furiosus were examined using circular dichroism (CD) and differential scanning calorimetry (DSC) at pH 2.3, where PCP-0SH exists in monomeric form. DSC showed a strong dependence of the shape and position of the unfolding profiles on the scan rate, suggesting the stability of PCP-0SH under kinetic control. On DSC timescales, even at a scan rate of 1 deg. C/hour, heat denaturation of PCP-0SH was non-equilibrium. However, over a long period of incubation of the heat-denatured PCP-0SH at pre-transition temperatures, it refolded completely, indicating reversibility with very slow relaxation kinetics. The rates of refolding of the heat-denatured PCP-0SH determined from the time-resolved DSC and CD spectroscopic progress curves were found to be similar within experimental error, confirming the mechanism of refolding to be a two-state process. The equilibrium established with a relaxation time of 5080 seconds (at t(m)=46.5 degrees C), which is unusually higher than the relaxation times observed for mesophilic and hyperthermophilic proteins. The long relaxation time may lead to the apparent irreversibility of an unfolding process occurring on the DSC experiment timescale. The refolding rate (9.8 x 10(-5) s(-1)) peaked near the t(m) (=46.5 degrees C), whereas the stability profile reached maxima (11.8 kJ mol(-1)) at 17 degrees C. The results clearly indicate the unusual mode of protein destabilization via a drastic decrease in the rate of folding at low pH and still maintaining a high activation energy barrier (284 kJ mol(-1)) for unfolding, which provides an effective kinetic advantage to unusually stable proteins from hyperthermophiles.

Authors+Show Affiliations

Institute for Protein Research, Osaka University, 3-2 Yamada-oka, Suita, Osaka 565-0871, Japan.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

11884137

Citation

Kaushik, Jai K., et al. "The Unusually Slow Relaxation Kinetics of the Folding-unfolding of Pyrrolidone Carboxyl Peptidase From a Hyperthermophile, Pyrococcus Furiosus." Journal of Molecular Biology, vol. 316, no. 4, 2002, pp. 991-1003.
Kaushik JK, Ogasahara K, Yutani K. The unusually slow relaxation kinetics of the folding-unfolding of pyrrolidone carboxyl peptidase from a hyperthermophile, Pyrococcus furiosus. J Mol Biol. 2002;316(4):991-1003.
Kaushik, J. K., Ogasahara, K., & Yutani, K. (2002). The unusually slow relaxation kinetics of the folding-unfolding of pyrrolidone carboxyl peptidase from a hyperthermophile, Pyrococcus furiosus. Journal of Molecular Biology, 316(4), 991-1003.
Kaushik JK, Ogasahara K, Yutani K. The Unusually Slow Relaxation Kinetics of the Folding-unfolding of Pyrrolidone Carboxyl Peptidase From a Hyperthermophile, Pyrococcus Furiosus. J Mol Biol. 2002 Mar 1;316(4):991-1003. PubMed PMID: 11884137.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - The unusually slow relaxation kinetics of the folding-unfolding of pyrrolidone carboxyl peptidase from a hyperthermophile, Pyrococcus furiosus. AU - Kaushik,Jai K, AU - Ogasahara,Kyoko, AU - Yutani,Katsuhide, PY - 2002/3/9/pubmed PY - 2002/4/4/medline PY - 2002/3/9/entrez SP - 991 EP - 1003 JF - Journal of molecular biology JO - J Mol Biol VL - 316 IS - 4 N2 - In order to understand the thermodynamic and kinetic basis of the intrinsic stability of proteins from hyperthermophiles, the folding-unfolding reactions of cysteine-free pyrrolidone carboxyl peptidase (Cys142/188Ser) (PCP-0SH) from Pyrococcus furiosus were examined using circular dichroism (CD) and differential scanning calorimetry (DSC) at pH 2.3, where PCP-0SH exists in monomeric form. DSC showed a strong dependence of the shape and position of the unfolding profiles on the scan rate, suggesting the stability of PCP-0SH under kinetic control. On DSC timescales, even at a scan rate of 1 deg. C/hour, heat denaturation of PCP-0SH was non-equilibrium. However, over a long period of incubation of the heat-denatured PCP-0SH at pre-transition temperatures, it refolded completely, indicating reversibility with very slow relaxation kinetics. The rates of refolding of the heat-denatured PCP-0SH determined from the time-resolved DSC and CD spectroscopic progress curves were found to be similar within experimental error, confirming the mechanism of refolding to be a two-state process. The equilibrium established with a relaxation time of 5080 seconds (at t(m)=46.5 degrees C), which is unusually higher than the relaxation times observed for mesophilic and hyperthermophilic proteins. The long relaxation time may lead to the apparent irreversibility of an unfolding process occurring on the DSC experiment timescale. The refolding rate (9.8 x 10(-5) s(-1)) peaked near the t(m) (=46.5 degrees C), whereas the stability profile reached maxima (11.8 kJ mol(-1)) at 17 degrees C. The results clearly indicate the unusual mode of protein destabilization via a drastic decrease in the rate of folding at low pH and still maintaining a high activation energy barrier (284 kJ mol(-1)) for unfolding, which provides an effective kinetic advantage to unusually stable proteins from hyperthermophiles. SN - 0022-2836 UR - https://www.unboundmedicine.com/medline/citation/11884137/The_unusually_slow_relaxation_kinetics_of_the_folding_unfolding_of_pyrrolidone_carboxyl_peptidase_from_a_hyperthermophile_Pyrococcus_furiosus_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0022283601953550 DB - PRIME DP - Unbound Medicine ER -