Partial destabilization of native structure
Hyperthermophile proteins do not display the phenomenon known as cold denaturation, which is seen in many mesophile proteins. We were intrigued by this and wished to explore how the stability of a hyperthermophile protein could be lowered sufficiently to cause it to begin to display some aspects of cold denaturation. We succeeded in doing this, and demonstrating it with TIM from Pyrococcusfuriosus, by using a combination of guanidium hydrochloride and high temperature (thermo-chemical denaturation) to destroy surface electrostatic interactions in P. furiosusTIM. We were delighted to observe not only that destruction of surface electrostatic interactions brings about cold denaturation in this protein, but also heat-renaturation, with the extents of cold-denaturation and heat-renaturation depending upon the extent to which sub-structures within the protein had been destabilized. Thus, we showed that hyperthermophile proteins are destabilized in multiple stages, and that each destabilized stage displays cold-denaturation and heat-renaturation without, however, ever being able to recover its native hyperthermophile structure completely after having been subjected to these. This is in keeping with the observation that hyperthermophile proteins are often unable to refold, becoming trapped in kinetic traps involving partially-folded states from which they cannot emerge. We think that the irreversibility of complete unfolding of such proteins owes to the necessity of their being folded during translation but, of course, we are yet to demonstrate this.