Temperature-reversible eruptions of vesicles in model membranes studied by NMR

Abstract

Deuterium (2H) and phosphorus (31P) nuclear magnetic resonance (NMR) and freeze-fracture electron microscopy were used to study spontaneous vesiculation in model membranes composed of POPC:POPS with or without cholesterol. The NMR spectra indicated the presence of a central isotropic line, the intensity of which is reversibly and linearly dependent upon temperature in the L alpha phase, with no hysteresis when cycling between higher and lower temperatures. Freeze-fracture microscopy showed small, apparently connected vesicles that were only present when the samples were frozen (for freeze-fracture) from an initial temperature of 40-60 degrees C, and absent when the samples are frozen from an initial temperature of 20 degrees C. Analysis of motional narrowing was consistent with the isotropic lines being due to lateral diffusion in (and tumbling of) small vesicles (diameters approximately 50 nm). These results were interpreted in terms of current theories of shape fluctuations in large unilamellar vesicles which predict that small daughter vesicles may spontaneously "erupt" from larger parent vesicles in order to expel the excess area created by thermal expansion of the bilayer surface at constant volume. Assuming that all the increased area due to increasing temperature is associated with the isotropic lines, the NMR results allowed a novel estimate of the coefficient of area expansion alpha A in multilamellar vesicles (MLVs) which is in good agreement with micromechanical measurements upon giant unilamellar vesicles of similar composition. Experiments performed on unilamellar vesicles, which had been placed upon glass beads, confirmed that alpha A determined in this way is unchanged compared with the MLV case. Addition of the highly positively charged (extrinsic) myelin basic protein (MBP) to a POPC:POPS system showed that membrane eruptions of the type described here occur in response to the presence of this protein.

NMR Determination of Protein Partitioning into Membrane Domains with Different Curvatures and Application to the Influenza M2 Peptide

“…A partial association of the isotropic phase with the lamellar phase is consistent with EM images showing M2-containing small vesicles budding off of large unilamellar vesicles (8). A similar example of protein-induced membrane budding is reported for the myelin basic protein, which also causes an isotropic peak in the 31 P NMR spectra, and whose cryoelectron microscopy images show small daughter vesicles pinching off of large parent vesicles (44).…”

The Effect of Ceramide on Phosphatidylcholine Membranes: A Deuterium NMR Study

“…The center of the spectrum at 38°C exhibits an isotropic peak having ϳ0.4% of the total intensity. This peak's intensity increases linearly with temperature, reaching ϳ1% at 69°C; thus we attribute it to a small amount of vesicle budding (Nezil et al, 1992). Fig.…”

The effects of phospholipid structure on the function of a calcium pump

“…Addition of cholesterol is known to increase the thickness of a lipid bilayer in the liquid crystalline phase [26], and addition of cholesterol can have a major effect on the incorporation of the peptides into phospholipid bilayers. Effects of cholesterol on incorporation of Pzz into bilayers of di(C14:I)PC or di(C18:1)PC are relatively small but for P16 in di(CIS:l)PC the effect of cholesterol is very much more marked, with a very low level of incorporation at a 1:l molar ratio of cholesterol to phospholipid [23].…”