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Three-Dimensional Dynamic Structure of the Liquid-Ordered Domain in Lipid Membranes as Examined by Pulse-EPR Oxygen Probing

Witold K. Subczynski ^*, Anna Wisniewska ^* , James S. Hyde ^* and Akihiro Kusumi 

^* Department of Biophysics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226; Department of Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland; Membrane Mechanisms Project, ICORP, Japan Science and Technology Agency, The Institute for Frontier Medical Sciences, Kyoto University, Shougoin, Kyoto 606-8507, Japan

Correspondence: Address reprint requests to Witold K. Subczynski, Dept. of Biophysics, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226. Tel.: 414-456-4038; Fax: 414-456-6512; E-mail: subczyn{at}mcw.edu.

Membranes made of dimyristoylphosphatidylcholine and cholesterol, one of the simplest paradigms for the study of liquid ordered-disordered phase separation, were investigated using a pulse-EPR spin-labeling method in which bimolecular collision of molecular oxygen with the nitroxide spin label is measured. This method allowed discrimination of liquid-ordered, liquid-disordered, and solid-ordered domains because the collision rates (OTP) differ in these domains. Furthermore, the oxygen transport parameter (OTP) profile across the bilayer provides unique information about the three-dimensional dynamic organization of the membrane domains. First, the OTP in the bilayer center in the liquid-ordered domain was comparable to that in the liquid-disordered domain without cholesterol, but the OTP near the membrane surface (up to carbon 9) was substantially smaller in the ordered domain, i.e., the cholesterol-based liquid-ordered domain is ordered only near the membrane surface, still retaining high levels of disorder in the bilayer center. This property may facilitate lateral mobility in ordered domains. Second, in the liquid-disordered domain, the domains with 5 mol % cholesterol exhibited higher OTP than those without cholesterol, everywhere across the membrane. Third, the transmembrane OTP profile in the liquid-ordered domain that contained 50 mol % cholesterol dramatically differed from that which contained 27 mol % cholesterol.