A conformationally isoformic thermophilic protein with high kinetic unfolding barriers.
The basis for the stability of thermophilic proteins is of fundamental interest for extremophile biology. We investigated the folding and unfolding processes of the homotetrameric Thermoanaerobacter brockii alcohol dehydrogenase (TBADH). TBADH subunits were 4.8 kcal/mol less stable towards guanidinium chloride (GdmCl) unfolding compared to urea, indicating ionic modulation of TBADH stability. Strongly denaturing conditions promoted mono-exponential unfolding kinetics with linear dependence on denaturant concentration. Here TBADH unfolded >40-fold slower when extrapolated from urea as compared to GdmCl unfolding. A marked unfolding hysteresis was shown when comparing refolding and unfolding in urea. An unusual biphasic unfolding trajectory with an exceptionally slow phase at intermediate concentrations of GdmCl and urea was also observed. We advocate that TBADH forms two distinctly different tetrameric isoforms, and likely an ensemble of native states. This unusual supramolecular folding behavior has been shown responsible for formation of amyloidotic yeast prion strains and can have functional importance for TBADH.
Mishra
R
R
IFM-Department of Chemistry, Linköping University, Linköping, Sweden.
Olofsson
L
L
Karlsson
M
M
Carlsson
U
U
Nicholls
I A
IA
Hammarström
P
P
D000426:Alcohol Dehydrogenase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D019281:Dimerization; D019791:Guanidine / Q000494:pharmacology; D007527:Isoenzymes; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D011328:Prions; D011487:Protein Conformation; D017510:Protein Folding; D020673:Protein Renaturation; D012441:Saccharomyces cerevisiae; D046968:Thermoanaerobacter / Q000201:enzymology* / Q000254:growth & development; D013816:Thermodynamics; D014508:Urea / Q000494:pharmacology
D003432:Cross-Linking Reagents; D007527:Isoenzymes; D011328:Prions; D014508:Urea; D000426:Alcohol Dehydrogenase; D019791:Guanidine