Cu2+Binding Modes of Recombinant α-Synuclein − Insights from EPR Spectroscopy
- 22 May 2008
- journal article
- research article
- Published by American Chemical Society (ACS) in Journal of the American Chemical Society
- Vol. 130 (24), 7766-7773
- https://doi.org/10.1021/ja800708x
Abstract
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−, NIm} or {NIm, 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.Keywords
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