Spin‐Label EPR on α‐Synuclein Reveals Differences in the Membrane Binding Affinity of the Two Antiparallel Helices

The putative function of the Parkinson's disease‐related protein α‐Synuclein (αS) is thought to involve membrane binding. Therefore, the interaction of αS with membranes composed of zwitterionic (POPC) and anionic (POPG) lipids was investigated through the mobility of spin labels attached to the protein. Differently labelled variants of αS were produced, containing a spin label at positions 9, 18 (both helix 1), 69, 90 (both helix 2), and 140 (C terminus). Protein binding to POPC/POPG vesicles for all but αS140 resulted in two mobility components with correlation times of 0.5 and 3 ns, for POPG mole fractions >0.4. Monitoring these components as a function of the POPG mole fraction revealed that at low negative‐charge densities helix 1 is more tightly bound than helix 2; this indicates a partially bound form of αS. Thus, the interaction of αS with membranes of low charge densities might be initiated at helix 1. The local binding information thus obtained gives a more differentiated picture of the affinity of αS to membranes. These findings contribute to our understanding of the details and structural consequences of αS–membrane interactions.

[1]  Ralf Langen,et al.  Structural Organization of α-Synuclein Fibrils Studied by Site-directed Spin Labeling* , 2003, Journal of Biological Chemistry.

[2]  A. Bax,et al.  Comparison of Structure and Dynamics of Micelle-bound Human α-Synuclein and Parkinson Disease Variants* , 2005, Journal of Biological Chemistry.

[3]  D. Marsh,et al.  Alpha-synuclein association with phosphatidylglycerol probed by lipid spin labels. , 2003, Biochemistry.

[4]  J. Freed,et al.  Inter-Helix Distances in Lysophospholipid Micelle-Bound α-Synuclein from Pulsed ESR Measurements , 2006 .

[5]  Richard Paylor,et al.  Synaptic Vesicle Depletion Correlates with Attenuated Synaptic Responses to Prolonged Repetitive Stimulation in Mice Lacking α-Synuclein , 2002, The Journal of Neuroscience.

[6]  Martina Huber,et al.  Distances between the b-subunits in the tether domain of F(0)F(1)-ATP synthase from E. coli. , 2005, Biochimica et biophysica acta.

[7]  D. Marsh,et al.  Association of α-synuclein and mutants with lipid membranes: spin-label ESR and polarized IR. , 2006 .

[8]  H. Khorana,et al.  Transmembrane protein structure: spin labeling of bacteriorhodopsin mutants. , 1990, Science.

[9]  Arthur Schweiger,et al.  EasySpin, a comprehensive software package for spectral simulation and analysis in EPR. , 2006, Journal of magnetic resonance.

[10]  W. Webb,et al.  Quantification of α-Synuclein Binding to Lipid Vesicles Using Fluorescence Correlation Spectroscopy , 2006 .

[11]  N. Hattori,et al.  A Combinatorial Code for the Interaction of α-Synuclein with Membranes* , 2005, Journal of Biological Chemistry.

[12]  Michel Goedert,et al.  Parkinsons Disease and other α-Synucleinopathies , 2001 .

[13]  Michel Goedert,et al.  Alpha-synuclein and neurodegenerative diseases , 2001, Nature Reviews Neuroscience.

[14]  Reinhard Jahn,et al.  A Broken α-Helix in Folded α-Synuclein* , 2003, The Journal of Biological Chemistry.

[15]  R. Langen,et al.  Side Chain-dependent Stacking Modulates Tau Filament Structure* , 2006, Journal of Biological Chemistry.

[16]  P. S. St George-Hyslop,et al.  Defective membrane interactions of familial Parkinson's disease mutant A30P α-synuclein , 2002 .

[17]  H. Khorana,et al.  Structural features of the C-terminal domain of bovine rhodopsin: a site-directed spin-labeling study. , 1999, Biochemistry.

[18]  David F. Clayton,et al.  Characterization of a novel protein regulated during the critical period for song learning in the zebra finch , 1995, Neuron.

[19]  S. Mammi,et al.  The 11-mer repeats of human alpha-synuclein in vesicle interactions and lipid composition discrimination: a cooperative role. , 2006, Biopolymers.

[20]  P. S. St George-Hyslop,et al.  α-Synuclein Membrane Interactions and Lipid Specificity* , 2000, The Journal of Biological Chemistry.

[21]  J. Feix,et al.  Guanidine hydrochloride unfolding of a transmembrane β‐strand in FepA using site‐directed spin labeling , 1998, Protein science : a publication of the Protein Society.

[22]  S. Mandel,et al.  Neurological mechanisms of green tea polyphenols in Alzheimer's and Parkinson's diseases. , 2004, The Journal of nutritional biochemistry.

[23]  R. Perrin,et al.  Interaction of human alpha-Synuclein and Parkinson's disease variants with phospholipids. Structural analysis using site-directed mutagenesis. , 2000, The Journal of biological chemistry.

[24]  K. Beyer.  Mechanistic aspects of Parkinson’s disease: α-synuclein and the biomembrane , 2007, Cell Biochemistry and Biophysics.

[25]  J. Fetrow,et al.  Kinetics and motional dynamics of spin-labeled yeast iso-1-cytochrome c: 1. Stopped-flow electron paramagnetic resonance as a probe for protein folding/unfolding of the C-terminal helix spin-labeled at cysteine 102. , 1997, Biochemistry.

[26]  Jack H Freed,et al.  Rigid body refinement of protein complexes with long-range distance restraints from pulsed dipolar ESR. , 2007, Methods in enzymology.

[27]  R. Langen,et al.  Spin labeling analysis of amyloids and other protein aggregates. , 2006, Methods in enzymology.

[28]  A. Jonas,et al.  Stabilization of α-Synuclein Secondary Structure upon Binding to Synthetic Membranes* , 1998, The Journal of Biological Chemistry.

[29]  S. Scarlata,et al.  Membrane binding and self-association of alpha-synucleins. , 2001, Biochemistry.

[30]  K. Hideg,et al.  Motion of spin-labeled side chains in T4 lysozyme. Correlation with protein structure and dynamics. , 1996, Biochemistry.

[31]  L. Serpell,et al.  Fiber diffraction of synthetic alpha-synuclein filaments shows amyloid-like cross-beta conformation. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[32]  Ralf Langen,et al.  Structure of membrane-bound α-synuclein studied by site-directed spin labeling , 2004 .

[33]  D. Eliezer,et al.  Helix periodicity, topology, and dynamics of membrane‐associated α‐Synuclein , 2005 .

[34]  M. L. Schmidt,et al.  α-Synuclein in Lewy bodies , 1997, Nature.

[35]  D. Eliezer,et al.  Conformational properties of alpha-synuclein in its free and lipid-associated states. , 2001, Journal of molecular biology.