The neuronal protein alpha-synuclein (α-syn) plays a central role in Parkinson's disease (PD). The pathological features of PD are the loss of dopaminergic neurons in the substantia nigra pars compacta and the presence of Lewy bodies (LBs). The C-terminal domain of α-syn is characterized by the presence of 15 acidic amino acids and all the 5 proline residues of the protein (P108, P117, P120, P128 and P138). The aggregation of this natively unfolded protein is accelerated in vitro by FK506 binding proteins (FKBPs) showing peptidyl-prolyl cis-trans isomerase (PPIase) activity. These proteins catalyze the cis-trans conformational change of the X-Pro peptide bond, often a rate limiting step in protein folding. The acceleration of the folding of α-syn by FKBPs may speed up disease associated aggregation. To further elucidate the role of the proline residues in the conformation and aggregation of α-syn, we constructed several mutants of α-syn where one or more proline residues are mutated to alanine via site-directed mutagenesis. For this purpose we produced and purified His-WT α-syn, a recombinant α-syn with a poly-histidine tag (6 His) and a linker and a number of Pro-to-Ala mutants. The aggregation kinetics of these mutants and His-WT α-syn were studied by turbidity, thioflavinT (ThT) fluorescence and CD measurements. We can conclude that mutation of the proline residues to alanine accelerates the aggregation kinetics of α-syn while all proline mutants formed fibrils similar to His-WT α-syn, as visualized with TEM. We also demonstrate that the accelerating effect of hFKBP12 is abolished by removing the proline residues from the C-terminus. Finally we show that the mutant of His α-syn with all five proline residues mutated to alanine, is more structured (more -helix) than His-WT α-syn, indicating the role of the Pro residues as potential helix breakers in the inhibitory conformation of the C-terminus.