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. Minimal Copper(II) Binding Sequence We have exploited a fluorescent amino acid, Trp, as a site-specific probe of Cu(II)-protein interaction. In particular, we found that Trp4 is the most responsive reporter of Cu(II), indicating a high-affinity N-terminal site with an apparent dissociation constant (100 nM, pH 7). (Lee JC, Gray HB, Winkler, JR, J. Am. Chem. Soc. 2008, 130, 6898-6899) We also demonstrated that the substitution of His50 with a Ser residue has little effect on the Cu(II)-affinity of the protein. More recently, we have identified the minimal amino acid sequence necessary to preserve Cu(II) affinity by employing a series of synthetic Trp-containing alpha-syn peptides (Jackson MS, Lee JC, Inorg. Chem. 2009, 48, 9303-9307). Surprisingly, the removal of more than 130 residues has relatively little effect on metal-protein interactions. Our data strongly indicate that only the first four residues are essential to preserve Cu(II) affinity. This peptide binds Cu(II) with a submicromolar dissociation constant comparable to the full length protein. These results clearly support our initial proposal that His50 is not required for N-terminal Cu(II) binding. Using mutant peptides (Lys-to-Arg), we also ruled out involvement of Lys 6 and/or Lys10 as metal ligands. To assess the participation of the alpha-amino moiety, the N-terminus was acetylated on all peptides. This modification completely disrupts Cu(II) coordination and assigns the alpha-amino group as the critical ligand. Our results provide direct evidence that for alpha-syn, a free N-terminal NH2 is required for Cu(II) binding. The finding that the alpha-amino terminus is crucial for Cu(II) binding in alpha-syn leads us to suggest a known motif in Cu(II)-polypeptide interactions: Cu(II) is anchored by the free amino-terminal nitrogen and chelates to adjacent backbone amides. Our data indicate that through post-translational modification of the N-terminus, copper-protein chemistry could be modulated in vivo. Effect of Dioxygen on Copper(II) Binding to alpha-Synuclein Using the fluorescent amino acid tryptophan, we have characterized the copper(II) binding of F4W alpha-syn in the presence and absence of dioxygen at neutral pH. Variations in Trp fluorescence indicate that copper(II) binding is enhanced by the presence of dioxygen, with the apparent dissociation constant changing from 100 nM (anaerobic) to 10 nM (aerobic). To investigate the possible role of methionine oxidation, complementary work focused on synthetic peptide models of the N-terminal Cu(II)-alpha-syn site, MDV(F/W) and M*DV(F/W), where M* = methionine sulfoxide. Furthermore, we employed circular dichroism (CD) spectroscopy to demonstrate that the phenyl-to-indole substitution does not alter copper(II) binding properties and to confirm the 1:1 metal-peptide binding stoichiometry. CD comparisons also revealed that Met1 oxidation does not affect the copper-peptide conformation and further suggested the possible existence of a novel Cu(II)-Trp/Phe (cation-pi) interaction (Lucas HR, Lee JC, J. Inorg. Biochem. 2010, 104, 245-249). Copper Reactivity of alpha-Synuclein Insights on the molecular details of soluble- and fibrillar-Cu-alpha-syn are gained through X-ray absorption spectroscopy. Our results indicate that the copper coordination environment (3-to-4 N/O ligands, average Cu-ligand distance 1.96 angstroms) exhibits little structural rearrangement upon amyloid formation in spite of the overall polypeptide conformational change from disordered-to-beta-sheet. Interestingly, we find that some population of Cu(II)-alpha-syn reduces to Cu(I)-alpha-syn in the absence of dioxygen. This autoreduction event appears diminished in the presence of O2 suggestive of preceding Cu(I)/dioxygen chemistry. Evidence for generation of reactive oxygen species is obtained by the observation of new emission features attributed to dityrosine crosslinks in fibrillar samples (Lucas, HR, DeBeer, S, Hong, M-S, Lee JC, J. Am. Chem. Soc. 2010, 132, 6636-6637).
| 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 |
