Membrane-bound alpha-synuclein forms an extended helix: long-distance pulsed ESR measurements using vesicles, bicelles, and rodlike micelles.
We apply pulsed dipolar ESR spectroscopy (Ku-band DEER) to elucidate the global conformation of the Parkinson's disease-associated protein, alpha-synuclein (alphaS) bound to small unilamellar phospholipid vesicles, rodlike SDS micelles, or lipid bicelles. By measuring distances as long as approximately 7 nm between introduced pairs of nitroxide spin labels, we show that distances are close to the expectations for a single continuous helix in all cases studied. In particular, we find distances of 7.5 nm between sites 24 and 72; 5.5 nm between sites 24 and 61; and 2 nm between sites 35 and 50. We conclude that alphaS does not retain a "hairpin" structure with two antiparallel helices, as is known to occur with spheroidal micelles, in agreement with our earlier finding that the protein's geometry is determined by the surface topology rather than being constrained by the interhelix linker. While the possibility of local helix discontinuities in the structure of membrane-bound alphaS remains, our data are more consistent with one intact helix. Importantly, we demonstrate that bicelles produce very similar results to liposomes, while offering a major improvement in experimentally accessible distance range and resolution, and thus are an excellent lipid membrane mimetic for the purpose of pulse dipolar ESR spectroscopy.
Georgieva
Elka R
ER
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, USA.
Ramlall
Trudy F
TF
Borbat
Peter P
PP
Freed
Jack H
JH
Eliezer
David
D
D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D008823:Micelles; D010743:Phospholipids / Q000737:chemistry* / Q000378:metabolism; D017433:Protein Structure, Secondary; D013572:Synaptic Vesicles / Q000737:chemistry / Q000378:metabolism; D053835:Unilamellar Liposomes / Q000737:chemistry; D051844:alpha-Synuclein / Q000737:chemistry* / Q000378:metabolism
D008823:Micelles; D010743:Phospholipids; D053835:Unilamellar Liposomes; D051844:alpha-Synuclein
J Biol Chem. 2003 Apr 25;278(17):15313-8
12586824
J Am Chem Soc. 2008 May 28;130(21):6690-1
18457394
Biochemistry. 2007 Apr 17;46(15):4499-509
17378587
Phys Chem Chem Phys. 2007 Apr 28;9(16):1895-910
17431518
J Neurosci. 2005 Nov 23;25(47):10913-21
16306404
Nat Struct Mol Biol. 2006 May;13(5):400-7
16622408
J Mol Biol. 2001 Apr 6;307(4):1061-73
11286556
Biochemistry. 2006 Jul 18;45(28):8453-65
16834319
J Am Chem Soc. 2002 May 15;124(19):5304-14
11996571
PLoS Biol. 2007 Oct;5(10):e271
17927448
Biochemistry. 2005 Jan 11;44(1):329-39
15628875
J Am Chem Soc. 2006 Aug 9;128(31):10004-5
16881616
Protein Sci. 2005 Apr;14(4):862-72
15741347
Biophys J. 2006 Jun 15;90(12):4692-700
16581836
Chem Commun (Camb). 2007 Jan 28;(4):377-9
17220976
Biochemistry. 2004 Apr 27;43(16):4810-8
15096050
Methods Enzymol. 2007;423:52-116
17609127
J Biol Chem. 2005 Mar 11;280(10):9595-603
15615727
J Magn Reson. 2008 Apr;191(2):202-18
18280189
Proc Natl Acad Sci U S A. 2004 Jun 1;101(22):8331-6
15155902
J Neurosci. 1988 Aug;8(8):2804-15
3411354
Q Rev Biophys. 2007 Feb;40(1):1-53
17565764
J Mol Biol. 2003 Jun 13;329(4):763-78
12787676