Isomers of human alpha-synuclein stabilized by disulfide bonds exhibit distinct structural and aggregative properties.
The discovery of three mutants in the -synuclein (alphaSyn) gene and the identification of alphaSyn as the major component of Lewy body have opened a new field for understanding the pathogenesis of Parkinson's disease (PD). AlphaSyn 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 alphaSyn (wt-alphaSyn) and two PD-associated alphaSyn mutants, A30P-alphaSyn and A53T-alphaSyn. This allows expression of three alphaSyn mutants, wt-alphaSyn(4C), A30P-alphaSyn(4C), and A53T-Syn(4C). Subsequent oxidative folding enables each alphaSyn(4C) mutant to form three partially stabilized two-disulfide isomers, designated as alphaSyn(2SS), that are amenable to further isolation and characterization. These alphaSyn mutants exhibit the following properties. (a) A30P-alphaSyn(4C) exhibits a lower folding flexibility than wt-alphaSyn(4C) and A53T-alphaSyn(4C). (b) All three alphaSyn(4C) mutants, like wt-alphaSyn, exhibit a predominant structure of random coil. However, wt-alphaSyn(2SS) adopts an alpha-helical conformation, whereas A30P-alphaSyn(2SS) and A53T-alphaSyn(2SS) take on significant beta-sheet structure. (c) A30P-alphaSyn(2SS) shows a stronger tendency to aggregate than A53T-alphaSyn(2SS) and wt-alphaSyn(2SS). (d) Three isolated isomers of wt-alphaSyn(2SS) exhibit a propensity for forming oligomers different yet enhanced versus that for wt-alphaSyn. These data together substantiate the notion that under physiological conditions, human alphaSyn exists as diverse conformational isomers which exhibit distinct propensities for aggregation and fibril formation.
Jiang
Chuantao
C
Research Center for Protein Chemistry, Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases and Department of Biochemistry and Molecular Biology, The University of Texas, Houston, Texas 77030, USA.
Chang
Jui-Yoa
JY
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002942:Circular Dichroism; D006801:Humans; D066298:In Vitro Techniques; D016631:Lewy Bodies / Q000378:metabolism; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010300:Parkinson Disease / Q000209:etiology / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051844:alpha-Synuclein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D046912:Multiprotein Complexes; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D051844:alpha-Synuclein