Biology Reference
In-Depth Information
After a correction of the protonation state, resulting structures are minimized using a
force field suitable for proteins (e.g., CHARMM;
Mackerell, Feig, & Brooks (2004)
)
and any of the available simulation software (e.g., NAMD and ACEMD). Finally, a
decent stereochemical quality of the minimized structures in terms of a good
distribution of
angles can be confirmed and visualized in the form of
Ramachandran plots by using a software such as PROCHECK (
Laskowski,
MacArthur, Moss, & Thornton, 1993
).
The final structures are now ready to be embedded into a lipid bilayer with a view
to simulate GPCR-membrane systems, a process described in the succeeding text.
C
and
F
4.2.2
Building and simulating GPCR-membrane complexes
As described in
Section 4.1.2
earlier, the structure of shortly equilibrated phospho-
lipid bilayers of customizable composition can be easily obtained using the
membrane-builder module of the CHARMM-GUI (
http://www.charmm-gui.org/
)
web interface. This module automatizes the process by generating the lipid bilayer,
embedding the protein into it, solvating and ionizing the membrane-protein complex,
and performing a short equilibration of the whole system. As for other membrane-
protein simulations, the size of the box is an important parameter since GPCRs need
sufficient space to evolve under periodic boundary conditions (i.e., a membrane
patch of about 94
94
˚
size is a typical choice for GPCR-membrane simulations).
Despite the CHARMM-GUI membrane builder can take care of the protein insertion
for us, which is useful to quickly build systems out of different initial starting struc-
tures, the receptor insertion can also be handled manually by using a molecular vi-
sualization software such as VMD. The latter approach gives more control over the
construction of the complete receptor-membrane system and enables to avoid arti-
facts such as certain undesirable interactions between molecules (i.e., lipid tails stuck
into the cholesterol ring).
Thus, in the first step, the modeled target GPCR is placed into the center of the
membrane patch. In the second step, those lipids that overlap with the receptor or that
are in close contact with the receptor (e.g., below 1
˚
distance from any protein
atoms) are deleted to avoid clashes during the simulation. This procedure generates
a hole that perfectly accommodates the target receptor. The receptor-membrane sys-
tem thereby obtained can then be solvated and neutralized by applying the solvate
and autoionize VMD plug-ins, which create a customized water box and assign
the type of ions needed to neutralize the system. As mentioned in
Section 4.1.2
,
the hydration level of the system is an important parameter that is frequently over-
looked. Similarly, the number and type of ions need to be carefully chosen at this step
in order to avoid unexpected results due to nonphysiological ion concentrations.
Once the GPCR is solvated by lipids, water, and ions, the system can be subjected
to a relaxation phase before starting the production phase. A typical relaxation phase
starts with a minimization procedure of 1000 steps before entering an equilibration
phase using an NPT ensemble with a target pressure equal to 1.01325 bar, a temper-
ature of 300 K, and a time step of 2 fs. In this relaxation phase, it is very important to
