title: Cu2+ binding modes of recombinant alpha-synuclein--insights from EPR spectroscopy.
abstract: The interaction of the small (140 amino acid) protein, alpha-synuclein (alphaS), with Cu(2+) 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 Cu(2+) binding modes in the full length protein remains comparatively less well characterized. This work examined the Cu(2+) binding of recombinant human alphaS using Electron Paramagnetic Resonance (EPR) spectroscopy. Wild type (wt) alphaS was shown to bind stoichiometric Cu(2+) via two N-terminal binding modes at physiological pH. An H50N mutation isolated one binding mode, whose g parallel, A parallel, and metal-ligand hyperfine parameters correlated well with a {NH2, N(-), beta-COO(-), H2O} mode previously identified in truncated model fragments. Electron spin-echo envelope modulation (ESEEM) studies of wt alphaS confirmed the second binding mode at pH 7.4 involved coordination of His50 and its g parallel and A parallel parameters correlated with either {NH2, N(-), beta-COO(-), N(Im)} or {N(Im), 2 N(-)} coordination observed in alphaS fragments. At pH 5.0, His50-anchored Cu(2+) binding was greatly diminished, while {NH2, N(-), beta-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 alphaS and was attributed to C-terminal binding centered on Asp121. In total, four Cu(2+) binding modes were identified within pH 5.0-7.4, providing a more comprehensive picture of the Cu(2+) binding properties of recombinant alphaS.
authors.affiliation: Department of Pathology, The University of Melbourne, Victoria 3010, Australia.
authors.forename: Simon C
authors.initials: SC
authors.lastname: Drew
authors.forename: Su Ling
authors.initials: SL
authors.lastname: Leong
authors.forename: Chi L L
authors.initials: CL
authors.lastname: Pham
authors.forename: Deborah J
authors.initials: DJ
authors.lastname: Tew
authors.forename: Colin L
authors.initials: CL
authors.lastname: Masters
authors.forename: Luke A
authors.initials: LA
authors.lastname: Miles
authors.forename: Roberto
authors.initials: R
authors.lastname: Cappai
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: Barnham
mesh_terms: D001665:Binding Sites; D003300:Copper / Q000737:chemistry* / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D006801:Humans; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010300:Parkinson Disease / Q000378:metabolism*; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051844:alpha-Synuclein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D051844:alpha-Synuclein; D003300:Copper