Andreas Bunge

Characterization of the Ternary Mixture of Sphingomyelin, POPC, and Cholesterol: Support for an Inhomogeneous Lipid Distribution at High Temperatures

A ternary lipid mixture of palmitoyl-oleoyl-phosphatidylcholine (POPC), palmitoyl-erythro-sphingosylphosphorylcholine (PSM), and cholesterol at a mixing ratio of 37.5:37.5:25 mol/mol/mol was characterized using fluorescence microscopy, (2)H NMR, and electron paramagnetic resonance spectroscopy. The synthetic PSM provides an excellent molecule for studying the molecular properties of raft phases. It shows a narrow phase transition at a temperature of 311 K and is commercially available with a perdeuterated sn-2 chain. Fluorescence microscopy shows that large inhomogeneities in the mixed membranes are observed in the coexistence region of liquid-ordered and liquid-disordered lipid phases. Above 310 K, no optically detectable phase separation was shown. Upon decrease in temperature, a redistribution of the cholesterol into large liquid-ordered PSM/cholesterol domains and depletion of cholesterol from liquid-disordered POPC domains was observed by (2)H NMR and electron paramagnetic resonance experiments. However, there is no complete segregation of the cholesterol into the liquid-ordered phase and also POPC-rich domains contain the sterol in the phase coexistence region. We further compared order parameters and packing properties of deuterated PSM or POPC in the raft mixture at 313 K, i.e., in the liquid crystalline phase state. PSM shows significantly larger (2)H NMR order parameters in the raft phase than POPC. This can be explained by an inhomogeneous interaction of cholesterol between the lipid species and the mutual influence of the phospholipids on each other. These observations point toward an inhomogeneous distribution of the lipids also in the liquid crystalline phase at 313 K. From the prerequisite that order parameters are identical in a completely homogeneously mixed membrane, we can determine a minimal microdomain size of 45-70 nm in PSM/POPC/cholesterol mixtures above the main phase transition of all lipids.

A reconstitution protocol for the in vitro folded human G protein-coupled Y2 receptor into lipid environment

Although highly resolved crystal structures of G protein-coupled receptors have become available within the last decade, the need for studying these molecules in their natural membrane environment, where the molecules are rather dynamic, has been widely appreciated. Solid-state NMR spectroscopy is an excellent method to study structure and dynamics of membrane proteins in their native lipid environment. We developed a reconstitution protocol for the uniformly (15)N labeled Y(2) receptor into a bicelle-like lipid structure with high yields suitable for NMR studies. Milligram quantities of target protein were expressed in Escherichia coli using an optimized fermentation process in defined medium yielding in over 10mg/L medium of purified Y(2) receptor solubilized in SDS micelles. The structural integrity of the receptor molecules was strongly increased through refolding and subsequent reconstitution into phospholipid membranes. Specific ligand binding to the integrated receptor was determined using radioligand affinity assay. Further, by NMR measurement a dispersion of the (15)N signals comparable to native rhodopsin was shown. The efficiency of the reconstitution could also be inferred from the fact that reasonable (13)C NMR spectra at natural abundance could be acquired.

Characterization of lipid bilayers adsorbed on spherical LbL-support

Structural and dynamic properties of membranes composed of phosphatidylcholine (PC) and phosphatidylserine (PS) on layer-by-layer (LbL) polyelectrolyte coated particles were investigated using solid-state nuclear magnetic resonance (NMR) and fluorescence methods. These spherically supported membranes showed structural, dynamic, and elastic properties similar to free-standing membranes as proved by 31P and 2H NMR. Small differences between behaviour of PC and PS on LbL support due to interaction with the polyelectrolyte were observed. Fluorescence lifetime imaging microscopy (FLIM) using 7-nitro-2-1,3-benzoxadiazol (NBD) labeled PC and PS showed a stronger impact of the outermost polyelectrolyte (PAH) on the fluorescence lifetimes of NBD-PS compared to NBD-PC. Although small defects in nm range allowing passage of Mn2+ to both layers of the membrane coat were present, a rather homogeneous coating observed by fluorescence microscopy, complete fluorescence recovery after photobleaching, and NMR results reveal that somewhat continuous lipid bilayers were formed around the LbL particles.

Biophysical Characterization of a New Phospholipid Analogue with a Spin-Labeled Unsaturated Fatty Acyl Chain

Spin-labeled analogs of phospholipids have been used widely to characterize the biophysical properties of membranes. We describe synthesis and application of a new spin-labeled phospholipid analog, SL-POPC. The advantage of this molecule is that the EPR active doxyl group is linked to an unsaturated fatty acyl chain different to saturated phospholipid analogs used so far. The need for those analogs arises from the fact that biological membranes contain unsaturated phospholipids to a large extent. The biophysical properties of SL-POPC in membranes were characterized using EPR and NMR spectroscopy and compared with those of the saturated spin-labeled phospholipid, SL-PSPC. To this end, POPC membranes were labeled with either analog to assess whether the spin-labeled counterpart SL-POPC mimics the membrane properties better than the often used SL-PSPC. The results show that SL-POPC and SL-PSPC explore different molecular environments of the bilayer, and that the type and degree of perturbation of bilayer caused by the label moiety also differs between both analogs. We found that SL-POPC is more appropriate to assess the versatile dynamics of POPC membranes than SL-PSPC.