Role of Protein-Water Interactions and Electrostatics in R-Synuclein Fibril Formation ² Larissa A. Munishkina, § Jeremy Henriques, § Vladimir N. Uversky,* ,§,‡ and Anthony L. Fink* ,§ Department of Chemistry and Biochemistry, UniVersity of California, Santa Cruz, California 95064, and Institute for Biological Instrumentation, Russian Academy of Sciences, Pushchino, Moscow Region 142292, Russia ReceiVed June 2, 2003; ReVised Manuscript ReceiVed January 14, 2004 ABSTRACT: Deposition of misfolded R-synuclein is a critical factor in several neurodegenerative disorders. Filamentous R-synuclein is the major component of Lewy bodies and Lewy neurites, the intracellular inclusions in the dopaminergic neurons of the substantia nigra, which are considered the pathological hallmark of Parkinson’s disease. We show here that anions induce partial folding of R-synuclein at neutral pH, forming a critical amyloidogenic intermediate, which leads to significant acceleration of the rate of fibrillation. The magnitude of the accelerating effect generally followed the position of the anions in the Hofmeister series, indicating a major role of protein-water-anion interactions in the process at salt concentrations above 10 mM. Below this concentration, electrostatic effects dominated in the mechanism of anion-induced fibrillation. The acceleration of fibrillation by anions was also dependent on the cation. Moderate concentrations of anions affected both the rates of nucleation and the elongation of R-synuclein fibrillation, primarily via their effect on the interaction of the protein with water. Parkinson’s disease (PD) 1 is a slowly progressive disease resulting from loss of dopaminergic neurons in the substantia nigra (1, 2). The aggregation of the presynaptic protein, R-synuclein, has been implicated in the formation of Lewy bodies and Lewy neurites, the intracellular inclusions characteristic of PD, and has been implicated in the etiology of the disease. Thus, although the molecular basis for the neurodegeneration in PD and other synucleinopathies is unknown as yet, the accumulation of R-synuclein-derived fibrillar material represents a potential link between protein misfolding and pathogenesis of these disorders. R-Synuclein is a small (140 amino acid residues), soluble, intracellular, highly conserved protein, of unknown function, that is abundant in various regions of the brain (3-6) and has been estimated to account for as much as 1% of the total protein in soluble cytosolic brain fractions (5). The amino acid sequence of R-synuclein is characterized by six imper- fect repeats (consensus KTKEGV) within the N-terminal half of the polypeptide, as well as by a highly acidic carboxyl- terminal region (6-8). Purified R-synuclein at neutral pH is characterized by a lack of ordered structure; i.e., it belongs to the family of natively unfolded or intrinsically unstructured proteins (9). R-Synuclein readily forms fibrils in vitro, with morphology and staining characteristics similar to those of fibrils extracted from disease-affected brain (9-17). The kinetics of fibrillation are consistent with a nucleation- dependent mechanism (11), in which the critical early stage involves the structural transformation into a partially folded intermediate (9). The rate of R-synuclein fibrillation is significantly ac- celerated by various environmental factors, including low pH and high temperature (9), heparin and other GAGs (18), metal cations (19, 20), polycations, including spermine, polyethyleneimine, poly-L-lysine, and poly-L-arginine (21), low concentrations of TMAO (22) simple and fluorinated alcohols (23), and certain pesticides (20, 24, 25). Accelerated fibrillation of R-synuclein in the presence of low concentra- tions of organic solvents (TMAO and alcohols) was shown to be due to the specific solvent-induced stabilization of an amyloidogenic partially folded intermediate (22, 23). A model has been suggested in which cations and pesticides interact with R-synuclein to bring about a conformational change to a partially folded state with a high propensity to aggregate (19, 20, 24, 25). The mechanisms of partially folded intermediate formation were suggested to be different for metal cations and pesticides. As the C-terminal region of R-synuclein (about 40 amino acids) is very rich in acidic residues and thus highly negatively charged at neutral pH, the resulting repulsive interactions are a major factor leading to the natively unfolded conformation of this protein. It has been suggested that the dominant effect of metal ions on R-synuclein conformational change and fibrillation is due to masking of the Coulombic charge-charge repulsion (19, 20). Similarly, polycations accelerate R-synuclein fibrillation by minimizing the electrostatic repulsion between negatively charged R-synuclein molecules, resulting in a high localized concentration of R-synuclein favoring fibrillation (21, 26). ² This research was supported by Grant RO1 NS39985 from the National Institutes of Health. * To whom correspondence should be addressed. Telephone: (831) 459-2744. Fax: (831) 459-2935. E-mail: uversky@hydrogen.ucsc.edu and fink@chemistry.ucsc.edu. § University of California, Santa Cruz. ‡ Russian Academy of Sciences. 1 Abbreviations: PD, Parkinson’s disease; AD, Alzheimer’s disease; LB, Lewy bodies; LN, Lewy neuritis; DLB, dementia with LB; LBVAD, LB variant of AD; MSA, multiple-system atrophy; WT, wild type; GAG, glycosaminoglycan; TMAO, trimethylamine-N-oxide; CD, circular dichroism; DLS, dynamic light scattering; ThT, thioflavin T; LDW, low-density water. 3289 Biochemistry 2004, 43, 3289-3300 10.1021/bi034938r CCC: $27.50 © 2004 American Chemical Society Published on Web 02/27/2004