When compared to #read more randurls[1|1|,|CHEM1|]# Syn-wt (~80 h), we observed that fibrillation was accelerated increasingly, depending on the number of negatively charged amino acid residues that were neutralized at the C-terminus. This finding served to isolate the effects of negative this site charge loss from the effects of polypeptide truncation, and show clearly that removal of negative charges from the C-terminal region of α-syn

results in an accelerated formation of fibrils, mainly through facilitating fibril nucleus formation. Inhibitors,research,lifescience,medical Figure 3 Fibril formation characteristics of the charge-free full-length α-syn mutants. Conditions were 1 mg/mL protein in 25 mmol/L Tris–HCl buffer, pH 7.5 at 37°C. Closed circles indicate Syn-wt; closed squares, Syn130-140CF; closed diamonds, … Role of tyrosine residue in the C-terminal region of α-syn on the fibril formation In the C-terminal region Inhibitors,research,lifescience,medical of α-syn, three tyrosine residues are also found at positions 125, 133, and 136. In order to understand the role of these tyrosine residues, we prepared several mutants where these tyrosines were selectively replaced with alanine residues, and examined the effects of these mutations on amyloid fibril formation. The

mutants constructed are grouped into single-residue substitution mutants (SynY125A, SynY133A, and SynY136A), double-substitution mutants (SynY125/133A, SynY125/136A, Inhibitors,research,lifescience,medical and SynY133/136A), and a mutant with all three tyrosines replaced (SynY125/133/136A). As shown in Figure

4, these mutants could be functionally regrouped into two groups, i.e., one group consisting of SynY125A, SynY133A, and SynY125/133A, which displayed a similar lag (nucleation) time (25–30 h) and Inhibitors,research,lifescience,medical fibril extension rates similar to that of Syn-wt. The other group included SynY136A, SynY125/136A, SynY133/136A, and SynY125/133/136A, with a prolonged nucleation time (60–70 h) and a slower fibril extension rate compared with Syn-wt. The common characteristic of members of the latter group was Inhibitors,research,lifescience,medical mutation of the tyrosine residue at position 136 to alanine. These results suggested strongly that Tyr136 plays a critical role in the amyloid fibril formation mechanism of α-syn. Figure 4 Fibril formation characteristics of various tyrosine substitution α-syn mutants. Conditions were 1 mg/ml protein in GSK-3 25 mmol/L Tris–HCl buffer, containing 1 mol/L NaCl, pH 7.5 at 37°C. Representations of symbols are explained in … In order to investigate further the role of Tyr136 in α-syn fibril formation, we replaced Tyr136 with various other amino acid residues. The mutants additionally prepared were substitutions to Trp, Phe, Leu, Ser, and Glu. Trp and Phe are hydrophobic and aromatic residues similar to Tyr, Leu is a residue with a hydrophobic aliphatic side chain, Ser is a hydrophilic residue without charge, and Glu represents a hydrophilic residue with charge.