Search

A persistent challenge in membrane biophysics has been to quantitatively predict how membrane physical properties change upon addition of new amphiphiles (e.g., lipids, alcohols, peptides, or proteins) in order to assess whether the changes are large enough to plausibly result in biological ramifications. Because of their roles as general anesthetics, n-alcohols are perhaps the best-studied amphiphiles of this class. When n-alcohols are added to model and cell membranes, changes in membrane parameters tend to be modest. One striking exception is found in the large decrease in liquid-liquid miscibility transition temperatures (Tmix) observed when short-chain n-alcohols are incorporated into giant plasma membrane vesicles (GPMVs). Coexisting liquid-ordered and liquid-disordered phases are observed at temperatures below Tmixin GPMVs as well as in giant unilamellar vesicles (GUVs) composed of ternary mixtures of a lipid with a low melting temperature, a lipid with a high melting temperature, and cholesterol. Here, we find that when GUVs of canonical ternary mixtures are formed in aqueous solutions of short-chain n-alcohols (n ≤ 10), Tmixincreases relative to GUVs in water. This shift is in the opposite direction from that reported for cell-derived GPMVs. The increase in Tmixis robust across GUVs of several types of lipids, ratios of lipids, types of short-chain n-alcohols, and concentrations of n-alcohols. However, as chain lengths of n-alcohols increase, nonmonotonic shifts in Tmixare observed. Alcohols with chain lengths of 10–14 carbons decrease Tmixin ternary GUVs of dioleoyl-PC/dipalmitoyl-PC/cholesterol, whereas 16 carbons increase Tmixagain. Gray et al. observed a similar influence of the length of n-alcohols on the direction of the shift in Tmix. These results are consistent with a scenario in which the relative partitioning of n-alcohols between liquid-ordered and liquid-disordered phases evolves as the chain length of the n-alcohol increases.

Insulin secretion depends on the Ca2+-regulated fusion of granules with the plasma membrane. A recent model of Ca2+-triggered exocytosis in secretory cells proposes that lipids in the plasma membrane couple the calcium sensor Syt1 to the membrane fusion machinery (Kiessling et al., 2018). Specifically, Ca2+-mediated binding of Syt1’s C2 domains to the cell membrane shifts the membrane-anchored SNARE syntaxin-1a to a more fusogenic conformation, straightening its juxtamembrane linker. To test this model in live cells and extend it to insulin secretion, we enriched INS1 cells with a panel of lipids with different acyl chain compositions. Fluorescence lifetime measurements demonstrate that cells with more disordered membranes show an increase in fusion efficiency, and vice versa. Experiments with granules purified from INS1 cells and recombinant SNARE proteins reconstituted in supported membranes confirmed that lipid acyl chain composition determines SNARE conformation and that lipid disordering correlates with increased fusion. Addition of Syt1’s C2AB domains significantly decreased lipid order in target membranes and increased SNARE-mediated fusion probability. Strikingly, Syt's action on both fusion and lipid order could be partially bypassed by artificially increasing unsaturated phosphatidylserines in the target membrane. Thus, plasma membrane lipids actively participate in coupling Ca2+/synaptotagmin-sensing to the SNARE fusion machinery in cells.

The affinity of single-pass transmembrane (TM) proteins for ordered membrane phases has been reported to depend on their lipidation, TM length, and lipid accessible surface area. In this work, the raft affinities of the TM domain of the linker for activation of T cells and its depalmitoylated variant are assessed using free energy simulations in a binary bilayer system composed of two laterally patched bilayers of ternary liquid ordered (Lo) and liquid disordered (Ld) phases. These phases are modeled by distinct compositions of distearoylphosphatidylcholine, palmitoyloleoylphosphatidylcholine (POPC), and cholesterol, and the simulations were carried out for 4.5 μs/window. Both peptides are shown to preferentially partition into the Ld phase in agreement with model membrane experiments and previous simulations on ternary lipid mixtures but not with measurements on giant plasma membrane vesicles where the Lo is slightly preferred. However, the 500 ns average relaxation time of lipid rearrangement around the peptide precluded a quantitative analysis of free energy differences arising from peptide palmitoylation and two different lipid compositions. When in the Lo phase, peptides reside in regions rich in POPC and interact preferentially with its unsaturated tail. Hence, the detailed substructure of the Lo phase is an important modulator of peptide partitioning, in addition to the inherent properties of the peptide.

Discoidal lipid-protein nanoparticles known as nanodiscs are widely used tools in structural and membrane biology. Amphipathic, synthetic copolymers have recently become an attractive alternative to membrane scaffold proteins for the formation of nanodiscs. Such copolymers can directly intercalate into, and form nanodiscs from, intact membranes without detergents. Although these copolymer nanodiscs can extract native membrane lipids, it remains unclear whether native membrane properties are also retained. To determine the extent to which bilayer lipid packing is retained in nanodiscs, we measured the behavior of packing-sensitive fluorescent dyes in various nanodisc preparations compared with intact lipid bilayers. We analyzed styrene-maleic acid (SMA), diisobutylene-maleic acid (DIBMA), and polymethacrylate (PMA) as nanodisc scaffolds at various copolymer-to-lipid ratios and temperatures. Measurements of Laurdan spectral shifts revealed that dimyristoyl-phosphatidylcholine (DMPC) nanodiscs had increased lipid headgroup packing compared with large unilamellar vesicles (LUVs) above the lipid melting temperature for all three copolymers. Similar effects were observed for DMPC nanodiscs stabilized by membrane scaffolding protein MSP1E1. Increased lipid headgroup packing was also observed when comparing nanodiscs with intact membranes composed of binary mixtures of 1-palmitoyl-2-oleoyl-phosphocholine (POPC) and di-palmitoyl-phosphocholine (DPPC), which show fluid-gel-phase coexistence. Similarly, Laurdan reported increased headgroup packing in nanodiscs for biomimetic mixtures containing cholesterol, most notable for relatively disordered membranes. The magnitudes of these ordering effects were not identical for the various copolymers, with SMA being the most and DIBMA being the least perturbing. Finally, nanodiscs derived from mammalian cell membranes showed similarly increased lipid headgroup packing. We conclude that nanodiscs generally do not completely retain the physical properties of intact membranes.