Association of alpha-synuclein and mutants with lipid membranes: spin-label ESR and polarized IR

Biochemistry. 2006 Mar 14;45(10):3386-95. doi: 10.1021/bi052344d.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Circular Dichroism
  • Electron Spin Resonance Spectroscopy / methods*
  • Humans
  • Lipid Bilayers / chemistry*
  • Lipid Bilayers / metabolism
  • Lipids / chemistry
  • Membranes / chemistry*
  • Membranes / metabolism
  • Phosphatidylcholines / chemistry
  • Phosphatidylglycerols
  • Protein Binding
  • Protein Conformation
  • Proteins / chemistry
  • Proteins / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Spectrophotometry, Infrared / methods*
  • Spin Labels
  • Titrimetry
  • alpha-Synuclein / chemistry*
  • alpha-Synuclein / genetics
  • alpha-Synuclein / metabolism

Substances

  • Lipid Bilayers
  • Lipids
  • Phosphatidylcholines
  • Phosphatidylglycerols
  • Proteins
  • Recombinant Proteins
  • Spin Labels
  • alpha-Synuclein