title: Association of alpha-synuclein and mutants with lipid membranes: spin-label ESR and polarized IR.
abstract: Alpha-synuclein is a presynaptic protein, the A53T and A30P mutants of which are linked independently to early-onset familial Parkinson's disease. The association of wild-type alpha-synuclein with lipid membranes was characterized previously by electron spin resonance (ESR) spectroscopy with spin-labeled lipids [Ramakrishnan, M., Jensen, P. H., and Marsh, D. (2003) Biochemistry 42, 12919-12926]. Here, we study the interaction of the A53T and A30P alpha-synuclein mutants and a truncated form that lacks the acidic C-terminal domain with phosphatidylglycerol bilayer membranes, using anionic phospholipid spin labels. The strength of the interaction with phosphatidylglycerol membranes lies in the order: wild type approximately truncated > A53T > A30P > fibrils approximately 0, and only the truncated form interacts with phosphatidylcholine membranes. The selectivity of the interaction of the mutant alpha-synucleins with different spin-labeled lipid species is reduced considerably, relative to the wild-type protein, whereas that of the truncated protein is increased. Polarized infrared (IR) spectroscopy is used to study the interactions of the wild-type and truncated proteins with aligned lipid membranes and additionally to characterize the fibrillar form. Wild-type alpha-synuclein is natively unfolded in solution and acquires secondary structure upon binding to membranes containing phosphatidylglycerol. Up to 30-40% of the amide I band intensity of the membrane-bound wild-type and truncated proteins is attributable to beta-sheet structure, at the surface densities used for IR spectroscopy. The remainder is alpha-helix and residual unordered structure. Fibrillar alpha-synuclein contains 62% antiparallel beta-sheet and is oriented on the substrate surface but does not interact with deposited lipid membranes. The beta-sheet secondary-structural elements of the wild-type and truncated proteins are partially oriented on the surface of membranes with which they interact.
authors.affiliation: Max-Planck-Institut für biophysikalische Chemie, Abt. Spektroskopie, 37070 Göttingen, Germany.
authors.forename: Muthu
authors.initials: M
authors.lastname: Ramakrishnan
authors.forename: Poul H
authors.initials: PH
authors.lastname: Jensen
authors.forename: Derek
authors.initials: D
authors.lastname: Marsh
mesh_terms: D002942:Circular Dichroism; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism; D008055:Lipids / Q000737:chemistry; D008566:Membranes / Q000737:chemistry* / Q000378:metabolism; D010713:Phosphatidylcholines / Q000737:chemistry; D010715:Phosphatidylglycerols; D011485:Protein Binding; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013055:Spectrophotometry, Infrared / Q000379:methods*; D013113:Spin Labels; D017186:Titrimetry; D051844:alpha-Synuclein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D008051:Lipid Bilayers; D008055:Lipids; D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols; D011506:Proteins; D011994:Recombinant Proteins; D013113:Spin Labels; D051844:alpha-Synuclein