Isomers of human α-synuclein stabilized by disulfide bonds exhibit distinct structural and aggregative properties

The discovery of three mutants in the alpha-synuclein (alpha Syn) gene and the identification of alpha Syn as the major component of Lewy body have opened a new field for understanding the pathogenesis of Parkinson's disease (PD). alpha Syn is a natively unfolded protein with unknown function and unspecified conformational heterogeneity. In this study, we introduce four Ser/Ala -> Cys mutations at positions 9, 42, 69, and 89 in human wild-type alpha Syn (wt-alpha Syn) and two PD-associated alpha Syn mutants, A30P-alpha Syn and A53T-alpha Syn. This allows expression of three alpha Syn mutants, wt-alpha Syn(4C), A30P-alpha Syn(4C), and A53T-alpha Syn(4C). Subsequent oxidative folding enables each alpha Syn(4C) mutant to form three partially stabilized two-disulfide isomers, designated as alpha Syn(2SS), that are amenable to further isolation and characterization. These alpha Syn mutants exhibit the following properties. (a) A30P-alpha Syn(4C) exhibits a lower folding flexibility than wt-alpha Syn(4C) and A53T-alpha Syn(4C). (b) All three alpha Syn(4C) mutants, like wt-alpha Syn, exhibit a predominant structure of random coil. However, wt-alpha Syn(2SS) adopts an alpha-helical conformation, whereas A30P-alpha Syn(2SS) and A53T-alpha Syn(2SS) take on significant beta-sheet structure. (c) A30P-alpha Syn(2SS) shows a stronger tendency to aggregate than A53T-alpha Syn(2SS) and wt-alpha Syn(2SS). (d) Three isolated isomers of wt-alpha Syn(2SS) exhibit a propensity for forming oligomers different yet enhanced versus that for wt-alpha Syn. These data together substantiate the notion that under physiological conditions, human alpha Syn exists as diverse conformational isomers which exhibit distinct propensities for aggregation and fibril formation.