SANS, FRET, and ESR Reveal

Abstract

Membrane raft size measurements are crucial to understanding the stability and functionality of rafts in cells. The challenge of accurately measuring raft size is evidenced by the disparate nanometer-to-micron sizes of coexisting liquid domains that have been reported for the biologically relevant model membrane system SM (sphingomyelin)/POPC (1-palmitoyl-2-oleoyl-sn -glycero-3-phosphocholine)/Chol (cholesterol). By combining three techniques with different spatial sensitivities, electron spin resonance (ESR), Forster resonance energy transfer (FRET), and small-angle neutron scattering (SANS), we have significantly narrowed the uncertainty in domain size estimates for bSM (porcine brain SM)/POPC/Chol mixtures. Compositional trends in ESR and FRET data indicate domains, while SANS reports complete miscibility, consistent with the presence of domains no larger than approx. 6 nm radius at 25C in bSM/POPC/Chol. Upon replacing the natural SM mixture with synthetic palmitoyl SM, SANS reports coexisting liquid domains. As shown in the figure, PSM/POPC/Chol = 39/39/22 exhibits enhanced scattering indicative of coexisting domains at 15 and 25C (circles, triangles). Minimal scattering at high temperature (squares, diamonds) indicates nearly random mixing at 45 and 55C.View Large Image | View Hi-Res Image | Download PowerPoint Slide