title: NMR determination of pKa values in α-synuclein.
abstract: The intrinsically unfolded protein α-synuclein has an N-terminal domain with seven imperfect KTKEGV sequence repeats and a C-terminal domain with a large proportion of acidic residues. We characterized pK(a) values for all 26 sites in the protein that ionize below pH 7 using 2D (1) H-(15) N HSQC and 3D C(CO)NH NMR experiments. The N-terminal domain shows systematically lowered pK(a) values, suggesting weak electrostatic interactions between acidic and basic residues in the KTKEGV repeats. By contrast, the C-terminal domain shows elevated pK(a) values due to electrostatic repulsion between like charges. The effects are smaller but persist at physiological salt concentrations. For α-synuclein in the membrane-like environment of sodium dodecylsulfate (SDS) micelles, we characterized the pK(a) of His50, a residue of particular interest since it is flanked within one turn of the α-helix structure by the Parkinson's disease-linked mutants E46K and A53T. The pK(a) of His50 is raised by 1.4 pH units in the micelle-bound state. Titrations of His50 in the micelle-bound states of the E46K and A53T mutants show that the pK(a) shift is primarily due to interactions between the histidine and the sulfate groups of SDS, with electrostatic interactions between His50 and Glu46 playing a much smaller role. Our results indicate that the pK(a) values of uncomplexed α-synuclein differ significantly from random coil model peptides even though the protein is intrinsically unfolded. Due to the long-range nature of electrostatic interactions, charged residues in the α-synuclein sequence may help nucleate the folding of the protein into an α-helical structure and confer protection from misfolding.
authors.affiliation: Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269-3125, USA.
authors.forename: Robyn L
authors.initials: RL
authors.lastname: Croke
authors.forename: Sharadrao M
authors.initials: SM
authors.lastname: Patil
authors.forename: Jason
authors.initials: J
authors.lastname: Quevreaux
authors.forename: Debra A
authors.initials: DA
authors.lastname: Kendall
authors.forename: Andrei T
authors.initials: AT
authors.lastname: Alexandrescu
mesh_terms: D000595:Amino Acid Sequence; D000682:Amyloid / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008823:Micelles; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D017510:Protein Folding; D012965:Sodium Chloride / Q000737:chemistry; D055672:Static Electricity; D051844:alpha-Synuclein / Q000737:chemistry* / Q000378:metabolism
chemical_list: D000682:Amyloid; D008823:Micelles; D051844:alpha-Synuclein; D012965:Sodium Chloride