1. and ​and2),2), we probed the contributions of electrostatic repulsion directly

by constructing substitution mutants where C-terminal Asp and/or Glu residues were altered to Asn residues (Fig. 3). From the results, it was revealed clearly that the acceleration of fibril formation, mainly fibril Protein Tyrosine Kinase inhibitor nucleus formation is due mainly to the elimination of negative charges, but not to decreases in polypeptide length of the C-terminal region. This result also agrees well with the finding that the fibril formation of α-syn is accelerated by charge shielding through addition of NaCl (Yagi et al. 2005), MgCl2, or spermine (Hoyer et al. 2004), or by lowering the pH (Hoyer et al. 2004; Cho et Inhibitors,research,lifescience,medical al. 2009; McClendon et al. 2009; Wu et al. 2009). The importance of the C-terminal 40 amino acids for fibril formation is also reported by Horvath et al. (2012) very recently. They observed an accelerated fibril formation of α-syn in the presence of a dihydro thiazolo ring fused 2-pyridone derivative, Inhibitors,research,lifescience,medical and found through NMR experiments that the compound interacts with amino Inhibitors,research,lifescience,medical acid residues 1–100, while residues 101–140 remained flexible in solution. This result demonstrates that masking the N-terminal region of the α-syn polypeptide results in conditions favorable for fibril formation, most likely by increasing accessibility to important portions

of the C-terminal region. As the fibril nucleus is stabilized by oligomerization, electrostatic repulsion between molecules may exert an inhibitory effect during fibril nucleus formation. Recently, we determined the fibril nucleus core peptide region of α-syn as the segment corresponding to Ala76–Lys96 (Yagi et al. 2010). When Inhibitors,research,lifescience,medical the 14 negative charges located between positions 104 and 139, which are relatively close to this nucleus core region, are removed by either deletion or mutation, intermolecular interactions of the fibril nucleus peptide regions would be favored, which Inhibitors,research,lifescience,medical in turn would accelerate fibril nucleus formation. Even under conditions similar

to the physiological salt concentration, the effects of negative charge were observed, as seen in Figure Terminal deoxynucleotidyl transferase 2b, suggesting that this electrostatic contribution is significant. In the C-terminal region of α-syn, we also find tyrosine residues at positions 125, 133, and 136. We have examined the relative contributions of these Tyr residues on fibril formation by mutating them to Ala residue(s) (Fig. 4), and found that Tyr136 was a critical element that promoted fibril formation. Simply changing Tyr136 to Ala was sufficient to significantly suppress both fibril nucleus formation (evidenced by the increased lag time) and fibril extension (seen by a decrease in the rate of fluorescence intensity increase). This Tyr could be replaced by Trp or Phe with minimal effects to the fibril formation mechanism, but not by Ser, Glu, or Leu (Fig. 5).