title: An electron paramagnetic resonance method for measuring the affinity of a spin-labeled analog of cholesterol for phospholipids.
abstract: Cholesterol (chol)-lipid interactions are thought to play an intrinsic role in determining lateral organization within cellular membranes. Steric compatibility of the rigid steroid moiety for ordered saturated chains contributes to the high affinity that holds chol and sphingomyelin together in lipid rafts whereas, conversely, poor affinity of the sterol for highly disordered polyunsaturated fatty acids (PUFAs) is hypothesized to drive the formation of PUFA-containing phospholipid domains depleted in chol. Here, we describe a novel method using electron paramagnetic resonance (EPR) to measure the relative affinity of chol for different phospholipids. We monitor the partitioning of 3β-doxyl-5α-cholestane (chlstn), a spin-labeled analog of chol, between large unilamellar vesicles (LUVs) and cyclodextrin (mβCD) through analysis of EPR spectra. Because the shape of the EPR spectrum for chlstn is sensitive to the very different tumbling rates of the two environments, the ratio of the population of chlstn in LUVs and mβCD can be determined directly from spectra. Partition coefficients (K(B)(A)) between lipids derived from our results for chlstn agree with values obtained for chol and confirm that decreased affinity for the sterol accompanies increasing acyl chain unsaturation. The virtue of this EPR method is that it provides a measure of chol binding that is quick, employs a commercially available probe and avoids the necessity for physical separation of LUVs and mβCD.
authors: Department of Physics, Indiana University-Purdue University Indianapolis, Indianapolis, IN, 46202-3273, USA.
authors: Justin A
authors: JA
authors: Williams
authors: Cynthia D
authors: CD
authors: Wassall
authors: Marvin D
authors: MD
authors: Kemple
authors: Stephen R
authors: SR
authors: Wassall
mesh_terms: D003497:Cyclic N-Oxides / Q000737:chemistry*; D003505:Cyclodextrins / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D010713:Phosphatidylcholines / Q000737:chemistry*; D013113:Spin Labels; D053835:Unilamellar Liposomes / Q000737:chemistry
chemical_list: D003497:Cyclic N-Oxides; D003505:Cyclodextrins; D010713:Phosphatidylcholines; D013113:Spin Labels; D053835:Unilamellar Liposomes; C002296:3-doxylcholestane
references: J Biol Chem. 2000 Jun 9;275(23):17221-4
references: 10770957
references: Biochim Biophys Acta. 2011 Mar;1808(3):727-32
references: 21167130
references: PLoS Comput Biol. 2011 Mar;7(3):e1002020
references: 21455285
references: Biochim Biophys Acta. 2010 May;1798(5):1008-13
references: 20044977
references: Biochemistry. 2004 Dec 14;43(49):15318-28
references: 15581344
references: Prog Lipid Res. 2002 Jan;41(1):66-97
references: 11694269
references: Chem Phys Lipids. 2013 Apr;169:113-23
references: 23291283
references: Biophys J. 2002 Dec;83(6):3408-15
references: 12496107
references: Science. 2010 Jan 1;327(5961):46-50
references: 20044567
references: Biochim Biophys Acta. 2009 Jan;1788(1):24-32
references: 19014904
references: Biochemistry. 1998 Dec 8;37(49):17299-308
references: 9860844
references: Biochim Biophys Acta. 1978 Dec 15;515(4):395-436
references: 215206
references: Biophys J. 2005 Aug;89(2):1109-19
references: 15923231
references: J Cell Sci. 1998 Jan;111 ( Pt 1):1-9
references: 9394007
references: J Lipid Res. 2006 Jul;47(7):1597-8
references: 16645198
references: Biochim Biophys Acta. 2003 Mar 10;1610(2):174-83
references: 12648772
references: FEBS Lett. 1999 May 21;451(2):197-202
references: 10371164
references: Biochemistry. 2010 Aug 3;49(30):6305-16
references: 20583817
references: J Biol Chem. 1986 Jun 25;261(18):8175-81
references: 3722148
references: Chem Phys Lipids. 2011 Nov;164(8):819-29
references: 21855534
references: J Am Chem Soc. 1972 Jun 28;94(13):4482-91
references: 4338554
references: Nature. 1997 Jun 5;387(6633):569-72
references: 9177342
references: J Biol Chem. 2003 Nov 14;278(46):45563-9
references: 12947110
references: Biophys J. 2001 Oct;81(4):2257-67
references: 11566796
references: Biophys J. 2008 Oct;95(8):3861-71
references: 18641061