Poster Presentation 2014 International Biophysics Congress

Small angle neutron scattering and NMR for probing flip flop in model membranes (#597)

Hanna MG Barriga 1 , Arwen II Tyler 1 , Oscar Ces 1 , Nicholas J Brooks 1 , Robert V Law 1 , John M Seddon 1
  1. Imperial College London, London, United Kingdom

Asymmetry across biological membranes is vital for a wide range of cellular functions1. Significant differences in lipid composition have been found between the inner and outer leaflets of bio-membranes, yet the rate of phospholipid flip-flop (movement of a lipid from one leaflet to the other) is currently very difficult to measure and estimates cover several orders of magnitude2. Critically, lipid modification to incorporate tags (for instance fluorescent groups) is likely to significantly affect flip-flop rates and so it is extremely important to develop chemical-label free methods of detecting asymmetry and measuring flip-flop rates.  We have used two complementary chemical-label free methods to probe flip-flop in lipid vesicles: small angle neutron scattering (SANS) and NMR.

Using contrast-variation SANS3 we have been able to systematically measure inter-vesicle lipid exchange rates and intra-vesicle flip-flop rates in model membrane systems.  These measurements have been performed over a range of temperatures and compositions, enabling us to decouple the inter-vesicle and intra-vesicle processes.

Lanthanide ions are able to both shift and broaden NMR signals given by phospholipids4.  By adding lanthanide ions to the external solution of unilamellar phospholipid vesicle samples and varying the lanthanide ion : lipid ratio we have been able to measure the flip-flop rate within the vesicle bilayers over a similar temperature range to the neutron scattering experiments.

By comparing the flip-flop rates obtained using these two different methods under directly comparable conditions we have begun to tackle long standing questions about the influence of experimental methods on measured flip-flop rates.

  1. G. van Meer, D.R.Voelker, G.W. Feigenson, Nat. Rev. Mol. Cell Biol, 2008, 9(2), 112-124, ‘Membrane lipids: where they are and how they behave’
  2. J.M. Sanderson, Mol. Membr. Biol, 2012, 29(5), 118-143, ‘Resolving the kinetics of lipid, protein and peptide diffusion in membranes’
  3. M. Nakano, M. Fukuda, T. Handa et al, J. Phys. Chem. B, 2009, 113, 6745-6748, ‘Flip-flop of phospholipids in vesicles: kinetic analysis with time resolved small angle neutron scattering’
  4. G.R.A. Hunt, L.R.H. Tipping, J. Inorg. Biochem, 1980, 12, 17-36, ‘Trans-bilayer pseudocontact shifts produced by lanthanide ions in the 1H and 31P NMR spectra of phospholipid vesicular membranes and their temperature variation’