Pulse EPR detection of lipid exchange between protein-rich raft and bulk domains in the membrane: Methodology development and its application to studies of influenza viral membrane

A pulse saturation-recovery electron paramagnetic resonance (EPR) method ha s been developed that allows estimation of the exchange rates of a spin-lab eled lipid between the bulk domain and the protein-rich membrane domain, in which the rate of collision between the spin label and molecular oxygen is reduced (slow-oxygen transport domain, or SLOT domain). It is based on the measurements of saturation-recovery signals of a lipid spin label as a fun ction of concentrations of both molecular oxygen and the spin label. Influe nza viral membrane, one of the simplest paradigms for the study of biomembr anes, showed the presence of two membrane domains with slow and fast collis ion rates with oxygen (a 16-fold difference) at 30 degreesC. The outbound r ate from and the inbound rate into the SLOT domain (or possibly the rate of the domain disintegration and formation) were estimated to be 7.7 x 10(4) and 4.6 x 10(4) s(-1), (15 mus residency time), respectively, indicating th at the SLOT domain is highly dynamic and that the entire SLOT domain repres ents about one-third of the membrane area. Because the oxygen transport rat e in the SLOT domain is a factor of two smaller than that in purple membran e, where bacteriorhodopsin is aggregated, we propose that the SLOT domain i n the viral membrane is the cholesterol-rich raft domain stabilized by the trimers of hemagglutinin and/or the tetramers of neuraminidase.