Cu2+ Binding Modes of Recombinant α-Synuclein − Insights from EPR Spectroscopy

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• 作者：Simon C. Drew ; Su Ling Leong ; Chi L. L. Pham ; Deborah J. Tew ; Colin L. Masters ; Luke A. Miles ; Roberto Cappai ; Kevin J. Barnham
• 刊名：Journal of the American Chemical Society
• 出版年：2008
• 出版时间：June 18, 2008
• 年：2008
• 卷：130
• 期：24
• 页码：7766 - 7773
• 全文大小：209K
• 年卷期：v.130,no.24(June 18, 2008)
• ISSN：1520-5126

文摘

The interaction of the small (140 amino acid) protein, α-synuclein (αS), with Cu²⁺ has been proposed to play a role in Parkinson’s disease (PD). While some insight from truncated model complexes has been gained, the nature of the corresponding Cu²⁺ binding modes in the full length protein remains comparatively less well characterized. This work examined the Cu²⁺ binding of recombinant human αS using Electron Paramagnetic Resonance (EPR) spectroscopy. Wild type (wt) αS was shown to bind stoichiometric Cu²⁺ via two N-terminal binding modes at physiological pH. An H50N mutation isolated one binding mode, whose g_∥, A_∥, and metal−ligand hyperfine parameters correlated well with a {NH₂, N⁻, β-COO⁻, H₂O} mode previously identified in truncated model fragments. Electron spin−echo envelope modulation (ESEEM) studies of wt αS confirmed the second binding mode at pH 7.4 involved coordination of His50 and its g_∥ and A_∥ parameters correlated with either {NH₂, N⁻, β-COO⁻, N_Im} or {N_Im, 2N⁻} coordination observed in αS fragments. At pH 5.0, His50-anchored Cu²⁺ binding was greatly diminished, while {NH₂, N⁻, β-COO⁻, H₂O} binding persisted in conjunction with another two binding modes. Metal−ligand hyperfine interactions from one of these indicated a 1N3O coordination sphere, which was ascribed to a {NH₂, CO} binding mode. The other was characterized by a spectrum similar to that previously observed for diethylpyrocarbonate-treated αS and was attributed to C-terminal binding centered on Asp121. In total, four Cu²⁺ binding modes were identified within pH 5.0−7.4, providing a more comprehensive picture of the Cu²⁺ binding properties of recombinant αS.