Folding intermediates of hyperthermophilic D-glyceraldehyde-3-phosphate dehydrogenase from Thermotoga maritima are trapped at low temperature.

D-Glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic eubacterium, Thermotoga maritima, is extremely thermostable showing a thermal transition beyond 105 degrees C. At low temperature, 'cold denaturation' becomes detectable only in the presence of destabilizing agents. Reconstitution after preceding denaturation depends on temperature. At 0 degree C, no significant recovery of activity is detectable, whereas between 30 and 100 degrees C reactivation reaches up to 85%. Shifting the temperature from low values to the range of optimum reconstitution releases the trapped intermediate in a fast reaction. Evidence from ultra-centrifugal analysis and far-UV circular dichroism proves the intermediate to be partially assembled to the tetramer, with most of its native secondary structure restored in a fast reaction. Fluorescence emission exhibits at least biphasic kinetics with the rate-limiting step(s) reflecting local adjustments of aromatic residues involved in tertiary contacts in the native state of the enzyme.