The leitmotif of this paper is that we identify yet another set of the key determinants of the extreme structural stability of rubredoxin from Pyrococcusfuriosus, a protein for which we had earlier performed and published a cold-denaturation study through destruction of surface electrostatic interactions by use of a thermo-chemical method of structural destabilization involving the electrolyte, and chaotrope, guanidium hydrochloride. We show that the protein is required to bind to iron early during its folding. If iron is not available, or not allowed to bind, the protein settles into a non-hyperthermostable structure of unremarkable structural stability – much like any ordinary thermophile-derived protein. On the other hand, if the protein binds to iron, the structure becomes ultra-hyperthermostable, causing rubredoxin to be the most stable protein known to man, at least as described in the literature. We devised a clever way of removing the iron from the folded, native, hyperthermostable form of rubredoxin, and showed that there is no change in the hyperthermostability. This indicated that iron binding was necessary to create hyperthermostability but not necessary for such hyperthermostabilty to be retained in the protein. Clearly, iron binding was somehow causing folding to occur differently, to create a different grade of structural stability. We used NMR spectroscopy to show that in the iron-bound, and iron-removed forms of rubredoxin, there is a different configuration of association of an aromatic cluster comprising six aromatic residues (two tryptophans, two tyrosines, and two phenylalanines), compared to the form of folded rubredoxin which was never allowed to bind iron during folding/refolding. Besides this core aromatic cluster, and any consequently enabled surface electrostatic interactions, rubredoxin in the iron-bound, iron-removed, and iron-never-bound states are indistinguishable by CD spectroscopy but distinguishable by fluorescence spectroscopy and by stability studies. We think that this is a really neat and detailed piece of work demonstrating how the differential configurations and associations of a small group of residues facilitated by metal binding can make an enormous difference in kinetic structural thermal stability in a protein.

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