Monomeric pores Water, glycerol, …

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

7. Constructing the Potential of Mean Force 4 trajectories v = 0.03, 0.015Å/ps k = 150 pN/Å

8. zp zc Constructing the Potential of Mean Force – cont. 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. Maltoporin OmpF GlpF AQP1 Assymetric structure; biological implication?

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. 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 Glycerol H H OH OH H H H OH H OH H H HO H OH H Cannot be verified by experimental measurements.

20. Complementarity glycerol molecule channel H H OH acceptor OH H H OH H hydrophobic H H donor H HO OH H H H HO HO 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. 2001 ACM Symposium on Interactive 3D Graphics, pp.191-194, ACM SIGGRAPH

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

24. Glycerol-Free GlpF

25. 18 water conducted In 4 monomers in 4 ns 1.125 water/monomer/ns Exp. = ~ 1-2 /ns Water permeation through GlpF 5 nanosecond Simulation 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: 0.4-0.8 e-5

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

30. H H H H H H H H H O O O O O O O O O H+ H H H H H H H H H H+ H+ H+ H+ H+ H+ H+ H+ H+ Proton transfer through water H+ H+ H+ H+ H+

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

32. Proton Blocking by a Global Orientation Mechanism