Superoxide dismutase folding/unfolding pathway: role of the metal ions in modulating structural and dynamical features.
The unfolding and refolding of a monomeric mutant of copper-zinc superoxide dismutase was investigated by NMR spectroscopy in the copper-reduced form and by using guanidinium chloride as denaturing agent. It is found that the protein gives rise to a series of intermediates at low guanidinium concentration and to a globular unfolded state at a guanidinium concentration higher than 3.5 M, which closely resembles a random coil structure, but with a high degree of compactness. At 1.9 M guanidinium, the intermediate and unfolded forms are present in equimolar ratio. The intermediate states show changes in the 1H and 15N chemical shifts with respect to the native protein. The perturbations on the signals occur at different GdmCl concentrations for different regions of the protein. The residues affected first are located in the loops and in the beta3 strand, followed by changes in the sheet formed by beta4, beta5, beta7, beta8 strands. The transition into the unfolded structure implies the detachment of the metal ions from the native coordination sites, even if non-specific interactions with the metal ions remain. R(1), R(2), [1H]-15N NOE, and CLEANEX experiments provide information on the mobility at the various stages showing how protein rigidity is lost during unfolding. The whole process is reversible. The oxidized species behaves in a similar way. The apo protein shows formation of 50% of the unfolded species at a guanidinium concentration of 0.4 M, thus demonstrating the importance of metal ions with respect to the unfolding process and protein structure stability. Hints to understand the whole folding process are obtained and discussed.
Assfalg
Michael
M
CERM, University of Florence, Via Luigi Sacconi 6, 50019 Sesto Fiorentino, Italy.
Banci
Lucia
L
Bertini
Ivano
I
Turano
Paola
P
Vasos
Paul R
PR
D002942:Circular Dichroism; D003300:Copper / Q000737:chemistry / Q000378:metabolism; D004058:Diffusion; D004220:Disulfides / Q000737:chemistry; D019791:Guanidine / Q000737:chemistry; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008670:Metals / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015032:Zinc / Q000737:chemistry / Q000378:metabolism
D004220:Disulfides; D008670:Metals; D003300:Copper; D013482:Superoxide Dismutase; D015032:Zinc; D019791:Guanidine