1. Aquaporins of known structure:
Tetrameric pore
Perhaps ions???
Monomeric pores
Water, glycerol, …
Aquaporins of known structure:
GlpF – E. coli glycerol channel (aquaglycerolporin)
AQP1 – Mammalian aquaporin-1 (pure water channel)

2. Revealing the Functional Role of Reentrant Loops

3. Single Glycerol per channel

4. We need to enforce an entire conduction event.
initial state
Note that glycerols moved, but not as extensively as earlier!
final (1ns) state
We need to enforce an entire conduction event.

5. Steered Molecular Dynamics
constant force
(250 pN)
constant velocity
(30 Å/ns)

6. Free Energy Calculation in SMD
SMD simulation
a non-equilibrium process
displacement
applied force
One needs to discount irreversible work
Jarzynski, PRL 1997
Hummer, PNAS, JCP 2001
Liphardt, et al., Science 2002
Free energy

7. Constructing the Potential of Mean Force
4 trajectories
v = 0.03, Å/ps
k = 150 pN/Å

8. Constructing the Potential of Mean Force – cont.zp zc
determined by minimizing the difference between (d) and (e)

9. Features of the Potential of Mean Force
Captures major features of the channel
The largest barrier  7.3 kcal/mol; exp.: 9.61.5 kcal/mol

10. Asymmetry of the Potential of Mean Force
phosphorylation
Cytoplasm
Periplasm
Asymmetric Profile in the Vestibules

11. Assymetric structure; biological implication?
OmpF
Maltoporin
GlpF
AQP1

12. Asymmetric structure of maltoporin

13. SMD Simulation of Glycerol Passage
Trajectory of glycerol pulled by constant force

14. Aquaporins’ Mechanisms of Selectivity
Protons, ions, and charged molecules cannot be transported.
Aquaglyceroporins (GlpF) transport linear sugars in a highly stereo-selective manner

15. Liposome Swelling Assay
carbohydrate
440 nm
H2O
carbohydrate +
H2O
shrinking
reswelling
scattered light
lipid vesicles
100 mM Ribitol
channel-containing proteoliposomes
Courtesy of Peter Nollert

16. Stereoselective Transport of Carbohydrates
Some stereoisomers show over tenfold difference in conductivity.
In vitro functional assay
Light scattering based transport assay, purified protein reconstituted into lipid vesicles
(black: membrane permeability, hatched: protein and membrane permeability)
GlpF specifically conducts linear polyalcohols, called alditols
Exclusion of circular carbohydrates
Stereoselectivity (ribitol vs D-arabitol)
stereoisomers

17. Channel Constriction GlpF + glycerol GlpF + water red < 2.3 Å
2.3 Å > green > 3.5 Å
blue > 3.5 Å
HOLE2: O. Smart et al., 1995

18. Selectivity filter

19. Evidence for Stereoselectivity of GlycerolH H OH H OH H H OH H OH H HO H H OH H
Cannot be verified by experimental measurements.

20. glycerol molecule channel
Complementarity
glycerol molecule channel H H OH H OH
acceptor H OH H
hydrophobic H
<Point to Glycerol # 3>
We were just talking about these two hydrogen bonds to G3
Between glycerol 3 and 2 there is a water molecule which suggests, that water is cotransported through the channel with glycerol in a one to one stochiometry.
Glycerol # 2 is bound in a similar way
Tripathic channel
Note that there is a hydrophilic side and a hydrophobic side in the channel which is complementary to the charge distribution pattern on the extended glycerol molecule. The channel and the glycerol molecule is tripathic, one hydrophobic side, an H-bond acceptor side and a H-bond donor site
The next slide illustrates a cut through G2 at these positions H
donor H OH HO H HO H HO H H OH HO H H H

21. Evidence for Stereoselectivity by Interactive Molecular Dynamics
Ribitol
Optimal hydrogen bonding and hydrophobic matching
Arabitol
10 times slower

22. Interactive Molecular Dynamics
Any PC/Workstation
Supports 3D force-feedback devices for interaction
VMD
NAMD
J. Stone, J. Gullingsrud, K. Schulten, and P. Grayson. A System for Interactive Molecular Dynamics Simulation ACM Symposium on Interactive 3D Graphics, pp , ACM SIGGRAPH

23. Results of Interactive Simulations
Restricted motion filter for both sugars
Pathways
An interesting position
Ribitol’s C-shaped conformation
Dipole reversal for ribitol at NPA

24. Glycerol-Free GlpF

25. 5 nanosecond Simulation 7-8 water molecules in each channel
Water permeation through GlpF
5 nanosecond Simulation
18 water conducted
In 4 monomers in 4 ns
1.125 water/monomer/ns
Exp. = ~ 1-2 /ns
7-8 water molecules in each channel

26. Correlated Movement of Water in the Channel
The single file of water molecules is maintained.

28. Diffusion of Water in the channel
One dimensional diffusion:
Experimental value for AQP1: e-5

29. Water Bipolar Configuration in Aquaporins
E.T. et al., Science 296, 525 (2002)

30. Proton transfer through water

31. Electrostatic Stabilization of Water Bipolar Arrangement
E.T. et al., Science 296, 525 (2002)

32. Proton Blocking by a Global Orientation Mechanism