Fig 1.
Methodology of membrane self-assembly.
(A) The self-assembly of evenly mixed lipids result in a membrane with symmetric leaflet. (B) two stacked boxes filled with different lipids for each intravesicular (IV) and cytoplasmic (CP) leaflet self-assembles in an asymmetric membrane. (C) The same method can be used in simulations of membrane-protein insertion by including the membrane protein in the initial set-up the self-assembly. The transmembrane domain (in purple) of the protein aligns itself with the hydrophobic core of the membrane.
Table 1.
Lipid composition of synaptic vesicle.
Fig 2.
Overlapping of IV and CP lipids that self-assemble to an asymmetric membrane.
IV lipids are mainly consisted of POPC and PPCS, and CP lipids are consisted of POPE and POPS. (A) The self- assembly with 0 nm overlap, in between IV and CP lipid boxes, separates into two membranes. The water molecules inserted (invisible) in between the gap of IV and CP prevents two boxes of lipids aggregating into a single membrane. But (B) in 0.5 nm overlapping, the center of IV + CP boxes is dense in lipids which prevents excessive water insertion leading to an asymmetric membrane with IV and CP leaflet. In both type of simulations the spontaneous formation occurred in 5 ns.
Table 2.
Lipid ratios of leaflet asymmetry in bilayer.
Fig 3.
Structural characteristics of the cholesterol free membranes.
(A) Potential energy profile over the simulation length for the asymmetric bilayer. (B) Box dimension, along x-axis, over time for asymmetric membrane (black) and symmetric membranes (see text) (IV-green, CP-red). (C) Partial density profiles of water (black) and various lipid moieties (HG—head group in red, PO4 in green, GL—glycerol backbone in blue, hydrophobic lipid tails in pink and terminal tail bead in brown) of the asymmetric bilayer along its normal (z-axis). The bilayer center is located at 0 nm. (D) Second-rank lipid order parameter for consecutive bonds of various lipids with respect to the bilayer normal. Data is plotted for all bonds involving headgroup (HG), phosphate (PO4), glycerol moieties (GL1, GL2) and the palmitoyl tail of the lipids for the asymmetric (upper panel) and IV symmetric (lower panel) bilayers.
Table 3.
Characteristics of asymmetric membrane systems.
Fig 4.
Lipid compositions in symmetric and asymmetric membrane.
(A) The membrane resulting from self-assembly of randomly mixed lipids exhibited a symmetric distribution for every type of phospholipid and cholesterol. (B) In membrane membranes resulting from asymmetric initial configuration, all lipid types were asymmetrically distributed. As in physiological membranes, PPCS and POPC were most abundant in the IV leaflet and POPS and POPE most abundant in the CP leaflet. Cholesterol was symmetrically distributed. (C) Frequency histograms of the distance of lipids’ center of mass to membrane’s center of mass (z = 0) shows the asymmetric lipid composition and also shows the presence of a small fraction of cholesterol located at the membrane center.
Fig 5.
Self-assembly of an asymmetric vesicle.
(A) A cut-away view of the vesicle at the start (0 ns) and at the end (135 ns) of the self-assembly simulation. The representation and coloring scheme is also shown. The head groups of different lipids are shown as spheres, the tails as sticks. (B) Snapshots of the entire vesicle illustrating the dynamics of the pores at different simulation times. Several pores formed within a few nanoseconds (2 ns). These pores coalesce to form a larger pore (25 ns) and finally the vesicle is sealed (60 ns).
Fig 6.
Equilibration in presence of “water-lined” pores.
(A) Vesicle showing the four “water-lined” pores (front, back, left, right) that were introduced into the self-assembled vesicle membrane to allow equilibration of lipid densities. The representation of vesicle lipids is same as in Fig 4, except for the wlipids (lipids with water particle type, yellow). (B) Cross section of one of the pores at the start (left) and the end (right) of a 200 ns equilibration. Note the tails of the wlipids (yellow) occupy the interior of the membrane at the start whereas they point away from the bilayer into the bulk solvent at the end. Also note the head groups of lipids that can be seen in the membrane interior, absent in left panel, showing the lipid exchange between the two leaflets. (C) The percent change in lipid composition of the IV leaflet of the vesicle during the equilibration. (D) Time evolution of APL of the CP and IV leaflets measured using Voronoi analysis on a patch of bilayer taken from the equilibrating vesicle.
Table 4.
Comparison of lipid number and ratios in vesicle and bilayer.
Fig 7.
Lipid distribution in an equilibrated vesicle.
Radial distribution function (RDF) g(r) for the phosphate beads of different lipid types as a function of distance from the center of mass of the entire vesicle. The vesicle size is calculated by following the inner and outer peaks in g(r) for the phosphate beads for all the phospholipids taken together (dashed line).
Fig 8.
Self-assembly of stx1A in an asymmetric bilayer.
(A) Snapshot of the insertion of stx1A in in the self- assembled bilayer (200 ns). The protein is shown in vdw representation and colored according to residue type. The lipids are shown in grey with phosphate group of phospholipids colored in tan and headgroup of cholesterol in purple. The PIP2 molecules are shown in orange. (B) Alignment of Stx1A (i) and surrounding PIP2 densities for Stx1A wt (ii) and 5RK/A mutant (iii) calculated from last 100 ns of three independent simulations. (C) Relative frequency of PIP2 interactions with residues 245 to 270 from the last 100 ns of 3 independent 200 ns simulations of Stx1A wt (filled bars) and 5RK/A mutant (open bars) (D) Trajectories of interaction of 3 individual PIP2 molecules (mol1 to mol3) with residues 245–270 of stx1A from one of the simulations.
Fig 9.
Self-assembly of the vesicle-protein system.
(A) Snapshot of vesicle at the start (left) and at the end (right) of self-assembly done along with twenty copies of syb2 molecules. To aid visualization, only the phosphate headgroups of the lipids and the backbone beads of the protein are shown. The phosphate headgroups of the outer leaflet are shown in yellow and the inner leaflet in magenta. The backbone beads of Syb2 copies that positioned themselves correctly are shown in green; three syb2 molecules that failed to position themselves correctly are colored red and blue. To illustrate the initial positioning of syb2, the left panel also shows theTrp90 in vdw representation and colored orange. The N- and C-term is also labeled for one of the syb2 molecule. (B) Comparison of the RDF g(r) of the lipid phosphate groups (black), cholesterol (red), Thr116 (green) and Trp90 (blue) at the start of the simulation (dotted) and during the last 50 ns of the self-assembly (solid). Note the small RDF peak of Thr116 (green solid) near the outer leaflet phosphate headgroup (red molecule in A) and the appearance of a Trp90 peak (blue solid) near inner leaflet phosphates (blue molecules in A), originating from misoriented syb2 molecules. (C) Evolution of the distance of backbone beads of Thr116 (top) and Trp90 (bottom) residues of all twenty protein molecules from the center of mass of the forming vesicle. The traces are colored following the coloring scheme of the molecules as shown in A.