Although recent work points to a genetic component to PD involving the accumulation and deposit of a neuronal protein, alpha-syn, the sporadic form of the disease is far more common and possibly connected to environmental factors that promote oxidative stress and aberrant redox-active metal metabolism. For example, selective accumulation of fibrils in dopaminergic neurons in PD has been attributed to the presence of easily oxidizable catechols that stimulate protein cross-links, as well as to increased iron concentration in Lewy bodies and copper in cerebrospinal fluid of PD patients. Furthermore, metal-enhanced oxidative oligomerization has been observed for alpha-syn in vitro, and, specific metal-protein interactions have been proposed to be critical in other neurodegenerative diseases involving amyloidogenic biomolecules such as amyloid beta-peptide (Alzheimers disease), prion protein (spongiform encephalopathies), and superoxide dismutase (amyotrophic lateral sclerosis). A difficult issue to resolve is whether metal ions perturb protein structures and thereby alter functions, or whether metal-protein complexes directly participate in the production of reactive oxygen species, or whether both mechanisms are at work. Copper(II) Enhances Membrane-bound alpha-Synuclein Helix Formation We have examined the effect of copper(II) on alpha-syn/lipid interactions and to test whether the phospholipid vesicles can modulate the copper(II) binding properties. Copper(II) is known to coordinate to soluble alpha-syn within the first four residues (MDVF) through the N-terminal amine and backbone amide chelation (Jackson MS, Lee JC Inorg. Chem. 2009, 48, 9303-9307). Importantly, the N-terminal region is also anticipated to interact electrostatically with cellular membranes through seven imperfect amino acid repeats (KXKEGV) in the primary amino acid sequence. We show that copper(II) binding to the N-terminus of alpha-syn affects its helical propensity and moreover, our results indicate that the copper(II) binding affinity of F4W alpha-syn is enhanced when the protein is membrane-bound. Interestingly, this work demonstrates that N-terminal membrane association is a dynamic process at least in regards to the copper-binding site penetrating the vesicle surface (Lucas HR, Lee JC, Metallomics 2011, 3, 280-283). Role of Asp2 in the Primary Copper (II) Binding Site in alpha-Synuclein Tryptophan fluorescence measurements involving mutant alpha-synucleins (F4W and F4W/H50S) and comparisons to synthetic N-terminal peptides suggest that the primary copper(II) binding site is localized in the first four residues. Moreover, the alpha-amino terminus is required for Cu(II) binding. Using site-directed mutagenesis (D2N/F4W and D2E/F4W), we are probing the nature of the copper(II)-D2 interaction showing that D2 is an unlikely copper(II) ligand. Insights on the molecular details of copper coordination environment also are gained through circular dichroism analyses and the use of synthetic peptide models. Coordination Properties of Cu(I) Bound alpha-Synuclein In prior work, structural information on copper coordination environment was obtained through X-ray absorption spectroscopy (Lucas, HR, DeBeer, S, Hong, M-S, Lee JC, J. Am. Chem. Soc. 2010, 132, 6636-6637). Particularly, we have characterized the local conformational changes in both soluble and fibrillar Cu-alpha-syn forms using Cu K-edge and extended X-ray fine structure absorption spectroscopy and have shown that there is Cu(I)/dioxygen reactivity during amyloid formation. Current work is focused on studying directly the ability of alpha-syn to bind copper(I), the physiologically relevant metal oxidation state.

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National Heart, Lung, and Blood Institute
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Lucas, Heather R; Lee, Jennifer C (2011) Copper(II) enhances membrane-bound ?-synuclein helix formation. Metallomics 3:280-3
Lucas, Heather R; Debeer, Serena; Hong, Myoung-Soon et al. (2010) Evidence for copper-dioxygen reactivity during alpha-synuclein fibril formation. J Am Chem Soc 132:6636-7
Lucas, Heather R; Lee, Jennifer C (2010) Effect of dioxygen on copper(II) binding to alpha-synuclein. J Inorg Biochem 104:245-9
Jackson, Mark S; Lee, Jennifer C (2009) Identification of the minimal copper(II)-binding alpha-synuclein sequence. Inorg Chem 48:9303-7