Iron-nucleated folding of a metalloprotein in high urea: resolution of metal binding and protein folding events.
Addition of iron salts to chaotrope-denatured aporubredoxin (apoRd) leads to nearly quantitative recovery of its single Fe(SCys)(4) site and native protein structure without significant dilution of the chaotrope. This "high-chaotrope" approach was used to examine iron binding and protein folding events using stopped-flow UV-vis absorption and CD spectroscopies. With a 100-fold molar excess of ferrous iron over denatured apoRd maintained in 5 M urea, the folded holoFe(III)Rd structure was recovered in >90% yield with a t(1/2) of <10 ms. More modest excesses of iron also gave nearly quantitative holoRd formation in 5 M urea but with chronological resolution of iron binding and protein folding events. The results indicate structural recovery in 5 M urea consists of the minimal sequence: (1) binding of ferrous iron to the unfolded apoRd, (2) rapid formation of a near-native ferrous Fe(SCys)(4) site within a protein having no detectable secondary structure, and (3) recovery of the ferrous Fe(SCys)(4) site chiral environment nearly concomitantly with (4) recovery of the native protein secondary structure. The rate of step 2 (and, by inference, step 1) was not saturated even at a 100-fold molar excess of iron. Analogous results obtained for Cys --> Ser iron ligand variants support formation of an unfolded-Fe(SCys)(3) complex between steps 1 and 2, which we propose is the key nucleation event that pulls together distal regions of the protein chain. These results show that folding of chaotrope-denatured apoRd is iron-nucleated and driven by extraordinarily rapid formation of the Fe(SCys)(4) site from an essentially random coil apoprotein. This high-chaotrope, multispectroscopy approach could clarify folding pathways of other [M(SCys)(3)]- or [M(SCys)(4)]-containing proteins.
Morleo
Anna
A
DISMA, University of Milan, Via G. Celoria 2, 20133 Milan, Italy.
Bonomi
Francesco
F
Iametti
Stefania
S
Huang
Victor W
VW
Kurtz
Donald M
DM
D001059:Apoproteins; D007501:Iron / Q000494:pharmacology*; D008667:Metalloproteins / Q000737:chemistry*; D008670:Metals; D011489:Protein Denaturation; D017510:Protein Folding / Q000187:drug effects*; D020673:Protein Renaturation; D012416:Rubredoxins / Q000737:chemistry*; D014508:Urea / Q000494:pharmacology
D001059:Apoproteins; D008667:Metalloproteins; D008670:Metals; D012416:Rubredoxins; D014508:Urea; D007501:Iron