Fibril extension rates of these two proteins were almost the same as Syn-wt. In contrast, SynY136E, SynY136S, SynY136A, and SynY136L showed CFTR inhibitor cost characteristics that were significantly different from Syn-wt, i.e., the nucleation times were prolonged and the fibril extension rates were clearly decreased compared to Syn-wt. In other words, aromatic residues at position 136 acted favorably toward both nucleus formation and fibril extension reactions, and hydrophilic or aliphatic residues at this

position disfavored this reaction. These findings supported our proposal that Tyr136 of α-syn plays an important Inhibitors,research,lifescience,medical role in the fibril formation, during both fibril nucleation and fibril extension. Figure 5 Fibril formation characteristics of various Tyr136 substitution α-syn mutants. Conditions were the same as described in the legend of Figure 4. Representations of symbols Inhibitors,research,lifescience,medical are explained in the figure. Standard error bars derived from at least three … Combining the Tyr136Ala mutation with charge neutralization in the C-terminal region As

shown above, the deletion of negative charges in the C-terminal region accelerated fibril formation of α-syn (Fig. 3), and the presence of tyrosine 136 in the same region promotes fibril formation by allowing nucleus formation (Fig. 4. and ​and5).5). In order to evaluate the relationship between these two modulating factors of fibril formation at Inhibitors,research,lifescience,medical neutral pH (near physiological conditions), we prepared Tyr136Ala mutants of Syn130-140CF and Syn119-140CF (hereafter called Syn130-140CF/Y136A and Syn119-140CF/Y136A, Inhibitors,research,lifescience,medical respectively; Table 1). To enhance our ability to detect fibril formation at pH 7.5 in the presence of 150 mmol/L NaCl, we used a multiwell plate reader for measurements, and also used the mixing function of the reader to promote fibril formation. It should be noted, thus, that this fibril formation experiment was performed under conditions different from Inhibitors,research,lifescience,medical other experiments. As shown in Figure 6, Syn130-140CF and Syn119-140CF

commenced fibril formation after a ~25- and ~5-h incubation, respectively, and both completed forming fibrils after a further 15-h incubation under these conditions. These reactions were accelerated compared to that (~45 h) of wild-type α-syn (see open circles in much Fig. 6), and a correlation was seen between the time necessary to complete fibril formation and the number of negative charges removed. When Tyr136 was changed to Ala in Syn119-140CF, fibril formation was greatly suppressed, indicating that the presence of Tyr138 was the predominant factor in the initiation of fibril formation that negates the effects of charge removal. This result is in overall agreement with the results shown in Figures 4 and ​and5.5. However, a most peculiar result was obtained when similar experiments were performed on the Syn130-140CF/Y136A mutant.