Phosphatidyl Glycerol

As a test for our parameters,
and to have a general understanding for how the three tested diterpenes
(Figure 1) would behave
in a membrane environment, we developed two distinct membrane models.
One model is identical with the yeast model used in prior terpenoid
permeation simulations.^{33 (link)} Each leaflet
for the yeast membrane was comprised of a 30:14:9:9:4:1 ratio for
phosphatidyl choline (PC), phosphatidyl ethanolamine (PE), phosphatidyl
inositol (PI), ergosterol, phosphatidyl serine (PS), and phosphatidic
acid (PA) headgroups, based on prior lipidomics work.^{44 (link)−47 (link)} This is consistent with the yeast composition for a previous study
on terpenoid permeation across yeast membranes.^{33 (link)} Similarly, the sorghum headgroup composition was a 35:25:23:12:6:2:2
ratio for PC, digalactosyl diacylglycerol (DGDG), monogalactosyl diacylglycerol
(MGDG), PE, sterols, phosphatidyl glycerol (PG), and PS, respectively,
based on available lipidomics data.^{48 (link)} The
sterol component within the sorghum membrane was split in a 3:2:1
ratio between palmitoyl sitosteryl glucoside, palmitoyl campesteryl
glucoside, and palmitoyl stigmasteryl glucoside, respectively.^{48 (link)} While it is well-known that real biological
membranes are generally asymmetric,^{49 (link)} available
lipidomics data were not collected with sufficient spatial resolution
to distinguish between the leaflets. Therefore, lipids and sterols
were symmetrically distributed in the two leaflets of the bilayer.
The fully detailed composition for both the model yeast and sorghum
membranes are presented in Table 1.
The membrane compositions for the yeast and sorghum
membranes from Table 1 were constructed
using the CHARMM-GUI web interface (Figure 2).^{50 (link)} Six total
simulation systems were generated, one for each possible diterpene-membrane
combination. Within each system, 20 copies for a given diterpene were
inserted in the system, ten initially above the membrane and ten below
the membrane using the TopoTools module within VMD.^{51 (link),52 (link)} The diterpenes placed in solution were displaced at 35 Å away
from the membrane center. Each system was solvated using the TIP3
water model through the solvate plugin within VMD.^{52 (link)} Counterions were added to neutralize the system and add
an extra 150 mM concentration of NaCl using the autoionize plugin
in VMD.^{52 (link)} Once complete, the yeast and
sorghum simulation systems were approximately 90 Å and
80 Å long along the membrane surface. Since sorghum has
larger glyco-lipid molecules, the simulation box was 115  Å
tall in the membrane normal dimension, while the yeast membrane was
100  Å tall. In total, the simulation systems contained
approximately 74,000 atoms and 67,000 atoms in yeast and sorghum simulation
systems, respectively, at a diterpene concentration of approximately
50 mM.
The protonation states assigned to the membrane
components were
consistent with pH 7. At this pH, abietic acid is generally deprotonated
and carries a formal negative charge, as the pK_a for abietic acid is around 4.7.^{53 (link)} However, prior experimental^{54 (link)} and computational
approaches^{22 (link),55 (link),56 (link)} have found that the neutral acid rather than the deprotonated carboxylate
is the major species that crosses the hydrophobic membrane interior.
Since the permeability coefficients for compounds with a carboxylate
group can be 8–12 orders of magnitude slower than the neutral
carboxylic acid,^22? the largest share of the permeants
will be the neutral form, as carboxylate forms of small molecules
are only 100–1000 times more abundant at pH 7. Thus, all simulations
carried out in this study use the neutral form for abietic acid, rather
than the conjugate base abietate, consistent with the structure shown
in Figure 1.