In this paper, we take a cue from our previous work showing cold-denaturation and heat-renaturation in Pyrococcus furious TIM through the unlocking of surface electrostatic interactions, and we extend it to the study of a much smaller protein of fewer than sixty residues, namely ‘rubredoxin’ from Pyrococcusfuriosus. We show that the rubredoxin from this hyperthermophile organism also shows stage-wise cold-denaturation and heat-renaturation, with many intermediate folded forms which constitute kinetically-trapped states which we call ‘Trishanku’ states. Of course, the rubredoxin was produced and folded in Escherichia coli like most of the proteins that we make and study, with the final structure formation to native structure occurring only when the produced and purified protein is first exposed to the temperature at which it had evolved to function in the organism for which it was evolved, close to the boiling point of water. We believe that this paper demonstrates that the theory of the kinetic control of protein folding dominates any thermodynamic control, in hyperthermophile proteins, most of which are unable to refold to native structure once they are unfolded.

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