Binding of a Single Zinc Ion to One Subunit of Copper-Zinc Superoxide Dismutase Apoprotein Substantially Influences the Structure and Stability of the Entire Homodimeric Protein

详细信息    查看全文

• 作者：Soshanna Zittin Potter ; Haining Zhu ; Bryan Francis Shaw ; Jorge A. Rodriguez ; Peter A. Doucette ; Se Hui Sohn ; Armando Durazo ; Kym F. Faull ; Edith Butler Gralla ; Aram M. Nersissian ; Joan Selverstone Vale
• 刊名：Journal of the American Chemical Society
• 出版年：2007
• 出版时间：April 18, 2007
• 年：2007
• 卷：129
• 期：15
• 页码：4575 - 4583
• 全文大小：354K
• 年卷期：v.129,no.15(April 18, 2007)
• ISSN：1520-5126

文摘

The thermodynamics of zinc binding to metal-free (apo) human and bovine copper-zincsuperoxide dismutases (SOD1) were measured using isothermal titration calorimetry. The apparentthermodynamics of zinc binding to the apoproteins were favorable (K_a > 10⁸ M^-1), with an observedstoichiometry of one zinc per homodimer. The change in heat capacity for the one-zinc binding event waslarge and negative (~ -650 cal mol^-1 K^-1), suggestive of significant structural changes to the protein uponzinc binding. We further characterized the one-zinc derivative by circular dichroism and determined thatthis derivative had nearly the same secondary structure as the two-zinc derivative and that both arestructurally distinct from the metal-free protein. In addition, we monitored the effect of zinc binding onhydrogen-deuterium exchange and accessibility of histidyl residues to modification by diethyl pyrocarbonateand observed that more than 50% protection was afforded by the binding of one zinc in both assays.Differential scanning calorimetry on the human SOD1 zinc derivatives also showed increased thermostabilityof the protein due to zinc binding. Further, the melting transitions observed for the one-zinc derivativeclosely resembled those of the two-zinc derivative. Finally, we observed that the quaternary structure ofthe protein is stabilized upon binding of one and two zinc ions in analytical ultracentrifugation experiments.Combined, these results suggest communication between the two monomers of SOD1 such that the bindingof one zinc ion per homodimer has a more profound effect on the homodimeric protein structure than thebinding of subsequent metal ions. The relevance of these findings to amyotrophic lateral sclerosis isdiscussed.