Membrane Proteins

1
Practicality of membrane protein simulations
Dr Phil Biggin
Dept. of Biochemistry University of
Oxford phil_at_biop.ox.ac.uk
2
Membrane Proteins Why?

• Ion channels, transporters, pumps, carriers,
  enzymes.
• Atomic level experimental information scarce
  (relatively)
• expression
• crystallization
• But key drug targets-

From Terstappen Reggiani, TIPS. 2001
3
Outline of Procedure
Obtain protein coordinates
Immerse in bilayer/mimetic
Solvate outside of membrane (and inside any
pore regions)
Add counterions (for correct concentration or to
satisfy electrostatic calculations)
Run simulation!
4
Protein Coordinates

• PDB - www.rcsb.org - X-ray/NMR
• Xray ? missing residues etc
• NMR ? which structure?
• Number of human membrane proteins at high
• resolution ZERO
• So have to start from homology model (eg from
  modeller)

5
Protein Coordinates
Starting from X-ray http//www.rcsb.org/
1BL8

• ? missing residues
• ? incomplete residues
• ? mutated residues
• ? oligomer state
• ? pH
• disulphides
• ? covalent linkages ions/solutes

Examine header
6
Prepare/Repair Structure
Graphically by hand (eg Quanta, Insight but these
cost ) Swissviewer is free but more limited.
Fix residues
Online automated versions (eg What-If server
http//www.cmbi.kun.nl1100/WIWWWI/ Will also
perform various stereochemical checks)
Prior knowledge
Oligomeric state
Macromolecular Structure Database http//www.ebi.a
c.uk/msd/

• Add polar hydrogens (Quanta, Insight but
  usually scripted in the other packages like
  pdb2gmx within the gromacs suite)

7
Prepare/Repair Structure

• pKa may be important protonation state of
  ionizable residues..
• Can do ad-hoc. Look at structure and assign by
  eye/distance the protonation state of a
  particular residue.
• Important for binding sites etc
• BUT can dramatically effect stability of protein
  in simulation
• For membrane proteins, situation is made more
  complex by presence of membrane

?78-90
Need different dielectric constants for each
region (interface region tricky still)
?2-6
?2-4
8
Alignment/Positioning in Box

• Things to consider-
• Existing experimental evidence
• The aromatic girdle

Z

• Energetic positioning-
• Assign a hydrophobicity value to each residue
  (many scales to chose from!)
• Calculate surface exposed area of each residue
• Decide on width of hydrophobic zone of membrane
  (30Å)
• Use Monte-Carlo to explore rigid-body movements
  across four degrees of freedom (3 rotational, 1
  translational along Z, the bilayer normal
• Lowest Energy position gives starting
  orientation with respect to box

9
Choice of mimetic
Full Bilayer Slowest but gain fullest information.
Octane Slab (speed but no detail)
Micelle Faster than bilayer. More and more NMR
data now.
Choice depends on what questions you want to
ask. For example is my homology model stable?
10
Inserting into Octane
YES
Fit new protein onto existing protein (and delete
existing protein)
Is system similar to existing one?
NO
minimize
Decide on box size and slab width
equilibrate
Solvate helix with new octane box
Run simulation!
Add water (and ions if needed)
Make slab thickness slightly more than what
you want as it will compress during equilibration.
11
Inserting in a Micelle
Easiest way is to build micelle around
protein May also have experimental data as to
the overall size estimate of the micelle.
Simply build by a script that relies on the
geometry of the system Solvate might
consider using an octahedral box for this system.
12
Insertion into Bilayer
KcsA is a membrane protein so solvation includes
bilayer, water and ions (sodium and chlorine for
example).
Add bilayer
Add water/ions
Cytosolic proteins immerse in box of
pre-equilibrated water and delete overlapping
(vdw spheres) molecules Sounds a lot easier
than it really is!
13
Protein into lipid

• Problem need to optimise interactions of lipids
  protein
• Method 1 Roux Woolf pack lipid around a
  protein
• Method 2 Faraldo-Gomez Smith - grow a hole
  in a pre-formed bilayer
• Method 3 Use genbox (gromacs) and run long
  equilibration
• Method 4 Use VMD plugins (designed primarily
  with NAMD in mind)

Change in Lipid Density
FhuA inserted in POPC bilayer
14
Insertion into Bilayer

• Possible Protocol (to be explored in the
  practical session)
• ? Obtain box of lipid.
• Put protein into same box dimensions.
• Use genbox to solvate the protein with that
  box of lipid.
• Add water with genbox.
• Delete waters in middle region of bilayer (perl
  script).
• Add any counter ions.
• Energy minimize.
• Few hundred picoseconds of restrained MD.
• Few nanoseconds of unrestrained MD (NPT).
• Check lipid properties.
• Perform production run (a few more nanoseconds).

15
Insertion into Bilayer
Things will equilibrate in a reasonably short
time (a few ns)
16
Solvation
Now add water either side (and anywhere else you
fancy) Adding bulk is easy - add lots of
small repeating boxes of water and delete
overlapping atoms (as implemented in for example
gromacs) For smaller pockets, cavities and
channels, you may need other more accurate
methods- Eg. MMC (a grand-canonical
monte-carlo approach) from Mihaly Mezei
Voidoo/Flood (from Uppsala) Solvate
(Grubmuller) NOTE May be better/easier to
solvate small cavities first prior to inserting
into the membrane.
Now add ions (number according to ionic strength)
Method 1 Random distribution Method 2
based on electrostatic potential
17
Ready to start?
First step is usually a minimization of
sorts. Strategy is ad-hoc really but work from
bits you trust backwards. E.G. Sample strategy
for membrane protein. ? Constrain protein atoms
minimize waters/lipids ? Constrain protein
atoms run MD for 200ps ? Constrain C? atoms
run MD for 200ps
18
Run it!
OK now you are in position to run free MD!
3

• Run to equilibrium.
• Use coordinate frames
• beyond that.
• 3. The more the merrier.

2
C? RMSD (Å)
1
Take frames from here
4
3
2
1
Time (ns)
19
Valid/stable simulation

• Lots of parameters to check but probably single
  most useful one is
• the area per lipid (describes molecular packing
  and describes degree of membrane fluidity).
• very sensitive to simulation details, considered
  to be a reliable criterion.
• Remember it depends what your question is,
  undulations across large patches require
  different timescales compared to water-headgroup
  interactions for example.

20
Parameters to consider in membrane simulations

• Periodic boundary conditions (PBCs) what shape
  box?
• Cubic, truncated octahedron, rhombic dodecahedron
• Amount of surrounding water typically more
  than 10Å margin
• Ensemble - NPT commonest, but there are others
  (NVE for example)
• Also constant surface tension simulations
• Pressure and temperature coupling
• E.g. Berendsen weak coupling versus
  Nosé-Hoover/Parrinello-Rahman
• Electrostatics Treatment
• Cut-off (artificial ordering?)
• Ewald methods, Particle Mesh Ewald (enhance
  periodicity)
• Reaction Field (ignore heterogenous nature of the
  membrane)
• Frequency of dump
• Large systems now (50,000-200,000 atoms) so files
  become large rapidly!
• Suggested dump every 5ps with currently sizes.

21
Insights from a recent Study

• A recent study systematically addressed some of
  the key issues in membrane simulations
  Methodological issues in lipid bilayer
  simulations. Anézo et al. J. Phys. Chem B.
  2003. 107. 9424-9433.
• Parameters investigated- electrostatic
  treatment (cutoff,PME,RF), cut-off radii, partial
  charge groupings, pressure coupling, timestep,
  size of system, force-field and amount of
  hydration water. (22 simulations some
  individually upto 150ns).
• Treatment of electrostatics has most impact on
  area but all three schemes can give correct area
  per lipid. Combination of this and force-field is
  what is important.
• Equilibration times of upto 25ns required for
  accurate assessment of properties such as area
  per lipid. Large area fluctuations occur on 10ns
  time scale.
• Area per lipid cannot tell you whether
  force-field or method is OK.
• But once area is correct, most others are usually
  OK (explains why so many different reports in the
  literature have bilayers with similar properties
• No difference with pressure coupling (though
  Berendsen might be preferred in equilibration as
  it damps oscillations more effectively)
• NO method is perfect! You make your choice!

22
Where do I get lipids from?
Far easier to start with ready-equilibrated
systems and insert protein into that
Scott Feller (wabash college)
http//persweb.wabash.edu/facstaff/fellers/ POPC,
DOPC, DPPC, SDPC Helmut Heller (München)
http//www.lrz-muenchen.de/heller/membrane/membra
ne.html POPC in different phases. Mikko
Karttunen (Helsinki) http//www.lce.hut.fi/researc
h/polymer/downloads.shtml DMTAP,DMPC,DPPC
Peter Tieleman (Calgary) http//moose.bio.ucalgary
.ca/Downloads/ DPC micelles, POPC, DMPC, DPPC
PLPC bilayers (topologies here as well). And
coming soon BioSimGrid lite http//www.biosimgr
id.org/ Various bilayers all with complete
topology and meta-data. More information about
this site in Fridays lecture.
23
What if I have strange topology?
Bonds and topology If have similar existing
topologies can adapt those. Can work out
manually (can be tiresome and boring!) Can use
PRODRG (Daan van Aalten) If using gromacs
someone might have already done it and uploaded
it! Charges Use similar atoms from similar
ligands Calculate from ab-initio (usual to use
partial charges that best reproduce
the molecular electrostatic potential (MEP) Vdw
Parameters Use similar atom types if
possible. Optimize to reproduce a range of
themodynamic properties (eg density)
24
Some references
D.M Hirst A computational approach to
chemistry Blackwell scientific publications
1990. A.R. Leach Molecular Modelling
Principles and Applications Longman Second ed.
2002. Gromacs manual _at_ http//www.gromacs.org M
ethodological issues in lipid bilayer
simulations. Anézo et al. J. Phys. Chem B.
2003. 107. 9424-9433. J.M. Haile Molecular
Dynamics Simulation Wiley 1997 Angwe Chemie
29 992 (1990)