Difference in the mechanisms of the cold and heat induced unfolding of thioredoxin h from Chlamydomonas reinhardtii: Spectroscopic and calorimetric studies

The thermodynamic stability and temperature induced structural changes of o xidized thioredoxin h from Chlamydomonas reinhardtii have been studied usin g differential scanning calorimetry (DSC), near- and far-UV circular dichro ism (CD), and fluorescence spectroscopies. At neutral pH, the heat induced unfolding of thioredoxin h is irreversible. The irreversibly unfolded prote in is unable to refold due to the formation of soluble high-order oligomers . In contrast, at acidic pH the heat induced unfolding of thioredoxin h is fully reversible and thus allows the thermodynamic stability of this protei n to be characterized. Analysis of the heat induced unfolding at acidic pH using calorimetric and spectroscopic methods shows that the heat induced de naturation of thioredoxin h can be well approximated by a two-state transit ion. The unfolding of thioredoxin h is accompanied by a large heat capacity change [6.0 +/- 1.0 kJ/(mol.K)], suggesting that at low pH a cold denatura tion should be observed at the above-freezing temperatures for this protein . All used methods (DSC, near-UV CD, far-UV CD, Trp fluorescence) do indeed show that thioredoxin h undergoes cold denaturation at pH <2.5. The cold d enaturation of thioredoxin h cannot, however, be fitted to a two-state mode l of unfolding. Furthermore, according to the far-UV CD, thioredoxin h is f ully unfolded at pH 2.0 and 0 degrees C, whereas the other three methods (n ear-UV CD, fluorescence, and DSC) indicate that under these conditions 20-3 0% of the protein molecules are still in the native state. Several alternat ive mechanisms explaining these results such as structural differences in t he heat and cold denatured state ensembles and the two-domain structure of thioredoxin h are discussed.