We are interested in why proteins from hyperthermophiles display very high kinetic structural stability. In this paper, we demonstrate in three ways, (i) by mutating residues, (ii) by changing pH, and (iii) by changing ionic strength, that when one disrupts salt-bridge electrostatic interactions on the surface of PyrococcusfuriosusTIM, the protein stops displaying its ultra-high kinetic structural stability and behaves like an ordinary thermostable protein from a thermophile organism, with much more rapid and facile unfolding at high temperatures close to the temperatures at which this enzyme has evolved to fold and function. This suggests that electrostatic interactions can be a key component of the ultra-stability of hyperthermophile proteins, with salt-bridges acting like ‘staples’ that tie together different structural elements, preventing them from undergoing facile cooperative unfolding and causing the consequent development of an abnormally-high kinetic thermal stability, owing largely to the slowing down on unfolding under conditions that are otherwise thermodynamically-destabilizing.

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