was constructed by placing two leaflets of short-tailed lipids at
the interface of water and 1,1-dichloroethane (DCLE), as described
in detail elsewhere.32 (link) The phosphatidylcholine
(PC) headgroup was selected for this study because of its relevance
to membrane composition of eukaryotic cells. The lipids used in the
membrane patch were constructed starting from palmitoyloleoylphosphatidylcholine
(POPC) molecule as a template and shortening its lipid tails to only
five carbons.32 (link) The HMMM membrane was then
assembled by use of Packmol software35 (link) by
constructing a DCLE box, with dimensions of 100 × 100 ×
10 Å3 and containing 5840 molecules of the organic
solvent, and placing 300 short-tailed PC lipids on its large faces,
with 150 lipids in each leaflet. The resulting structure was then
solvated with water by use of the SOLVATE plugin of VMD,36 yielding a system of ∼66 000 atoms.
The solvated membrane mimetic system was energy-minimized for 10 000
steps and simulated for 2 ns, by use of an NPnAT ensemble with constant area, and with a target normal pressure
and temperature of 1.0 atm and 310 K, respectively. A constant area
of 11 236 Å2 (106 × 106 Å2) was employed, yielding an area of ∼75 Å2/lipid (AL), which is ∼8% higher
than the experimental AL for POPC.37 (link) This was done to account for the area of the
membrane that would be occupied by the protein upon its insertion.
On the basis of our experience with several other peripheral proteins,
a mild increase (5–8%) in the area can significantly accelerate
the process. The resulting membrane was employed in all subsequent
simulations of membrane binding and dynamics of CYP3A4.