Journal
FEBS OPEN BIO
Volume 5, Issue -, Pages 56-63Publisher
WILEY
DOI: 10.1016/j.fob.2014.12.003
Keywords
NMR; SOD1; Folding; Metal-binding
Categories
Funding
- Swedish Research Council [621-2011-5964]
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The stability and structural properties of the metalloprotein superoxide dismutase 1 (SOD1) are found to depend critically on metal ions. Native SOD1 monomers coordinate one structural Zn2+ and one redox-active Cu2+/1+ to the active site. To do this, the Zn2+ ions need to interact with the SOD1 protein on the denatured side of the folding barrier, prior to the formation of the folding nucleus. In this study, we have examined at residue level the nature of this early Zn2+ binding by NMR studies on the urea denatured-state of SOD1. Nearly complete backbone chemical shift assignments were obtained in 9 M urea at physiological pH, conditions at which NMR studies are scarce. Our results demonstrate that SOD1 is predominantly unstructured under these conditions. Chemical-shift changes upon Zn2+ titration show that denatured SOD1 retains a significant affinity to Zn2+ ions, even in 9 M urea. However, the Zn2+ interactions are not limited to the native metal-binding ligands in the two binding sites, but are seen for all His residues. Moreover, the native Cu2+/1+ ligand H46 seems not to bind as well as the other His residues, while the nearby non-native H43 does bind, indicating that the binding geometry is relaxed. The result suggests that the Zn2+-binding observed to catalyze folding of SOD1 in physiological buffer is initiated by diffuse, non-specific coordination to the coil, which subsequently funnels by ligand exchange into the native coordination geometry of the folded monomer. Altogether, this diffuse binding is a result with fundamental implications for folding of metalloproteins in general. (C) 2015 The Authors. Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.
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