Rubredoxin variant folds without iron

Pyroccocus furiosus rubredoxin (PFRD), like most studied hyperthermophilic proteins, does not undergo reversible folding. The irreversibility of foldi ng is thought to involve PFRD's iron-binding site. Here we report a PFRD va riant (PFRD-XC4) whose iron binding site was redesigned to eliminate iron b inding using a computational design algorithm. PFRD-XC4 folds without iron and exhibits reversible folding with a melting temperature of 82 degrees C, a thermodynamic stability of 3.2 kcal mol(-1) at 1 degrees C, and NMR chem ical shifts similar to that of the wild-type protein. This variant should p rovide a tractable model system for studying the thermodynamic origins of p rotein hyperthermostability.