A step toward understanding the folding mechanism of bovine adrenodoxin.
The iron-sulfur clusters of iron-sulfur proteins are not only essential for the structure and function but they also seem to play an important role in the folding process of these proteins. So far, no data on reversible unfolding/refolding of iron-sulfur proteins under aerobic conditions have been reported. We found appropriate conditions, which might also be applicable for other iron-sulfur proteins, for reversible unfolding/refolding of bovine adrenodoxin (Adx) that prevent cluster decomposition during the unfolding process. The unfolding/refolding studies have been performed under aerobic conditions using fluorescence measurements (with mutant Y82W of Adx, providing a sensitive internal probe), absorption, and circular dichroism (CD) spectroscopy as well as activity measurements. Without protecting reagent, adrenodoxin becomes an apoprotein upon denaturation which is an irreversible process with respect to cluster rebinding. However, reversibility of unfolding/refolding can be observed after protein denaturation in the presence of dithiothreitol (DTT). Upon removal of the denaturant, we regained 65, 63, and 64% refolding from CD, fluorescence, and activity measurements, respectively. In the case of thermal denaturation, the percentage of refolding is about 60% according to CD measurements. DTT appears to stabilize the [2Fe-2S] cluster and prevents its decomposition during aerobic unfolding, providing thereby the means of correct refolding of the protein.
Bera
A K
AK
Fachbereich 12 Pharmazie und Umwelttechnologie, Fachrichtung 12.4 Biochemie, Universität des Saarlandes, Im Stadtwald, Saarbrücken, Germany.
Grinberg
A
A
Bernhardt
R
R
D000325:Adrenodoxin / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D004229:Dithiothreitol / Q000494:pharmacology; D004579:Electron Transport / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D013050:Spectrometry, Fluorescence
D000325:Adrenodoxin; D004229:Dithiothreitol