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Absence of kinetic thermal stabilization in a hyperthermophile rubredoxin indicated by 40 microsecond folding in the presence of irreversible denaturation
Author(s) -
LeMaster David M.,
Tang Jianzhong,
Hernández Griselda
Publication year - 2004
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.20181
Subject(s) - rubredoxin , chemistry , pyrococcus furiosus , crystallography , hyperthermophile , folding (dsp implementation) , protein folding , kinetics , denaturation (fissile materials) , biochemistry , physics , quantum mechanics , nuclear chemistry , electrical engineering , archaea , gene , engineering
The striking kinetic stability of many proteins derived from hyperthermophilic organisms has led to the proposal that such stability may result from a heightened activation barrier for unfolding independent of a corresponding increase in the thermodynamic stability. This in turn implies a corresponding retardation of the folding reaction. A commonly cited model for kinetic thermal stabilization is the rubredoxin from Pyrococcus furiosus ( Pf ), which exhibits an irreversible denaturation lifetime at 100°C of nearly a week. Utilizing protein resonances shifted well outside of the random coil chemical shift envelope, nuclear magnetic resonance (NMR) chemical exchange measurements on Pf rubredoxin as well as on the mesophile Clostridium pasteurianum ( Cp ) rubredoxin demonstrate reversible thermal transition temperatures of 144°C (137°C for the N‐terminal modified A2K variant) and 104°C, respectively, with similar (un)folding rates of ≈25,000 s −1 , only modestly slower than the diffusion controlled rate. The absence of a substantial activation barrier to rubredoxin folding as well as the similar folding kinetics of the mesophile protein indicate that kinetic stabilization has not been utilized by the hyperthermophile rubredoxin in achieving its extreme thermal stability. The two‐state folding kinetics observed for Pf rubredoxin contradict a previous assertion of multiphasic folding based on hydrogen exchange data extrapolated to an estimated midpoint of transition temperature ( T m ) of nearly 200°C. This discrepancy is resolved by the observation that the base‐catalyzed hydrogen exchange of the model dipeptide (N‐acetyl‐L‐cysteine‐N‐methylamide) 4 ‐Cd 2+ is 23‐fold slower than that of the free cysteine model dipeptide used to normalize the Pf rubredoxin hydrogen exchange data. Proteins 2004. © 2004 Wiley‐Liss, Inc.