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Keeping Aquaporin Channels Constitutively Open for Biotechnology Applications and more

Keeping Aquaporin Channels Constitutively Open for Biotechnology Applications and more. MEMPHYS, Center for Bio-Membrane Physics, Center of Excellence funded by The Danish National Research Foundation Southern Denmark University, Odense, Denmark. Periplasm. Cytoplasm.

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Keeping Aquaporin Channels Constitutively Open for Biotechnology Applications and more

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  1. Keeping Aquaporin Channels Constitutively Openfor Biotechnology Applications and more MEMPHYS, Center for Bio-Membrane Physics, Center of Excellence funded byThe Danish National Research Foundation Southern Denmark University, Odense, Denmark

  2. Periplasm Cytoplasm Aquaporin: the water pore Transmembrane water transporters Peter Agre, JHU 2003 Nobel Prize in Chemistry 50%

  3. Aquaporin • 109 water molecules per second • Fast • Very water selective • Pure • Bidirectional, passive • Mechanically/Osmotically driven transport • Robust protein Can be used to filter water in industrial applications?

  4. Laboratory testing Laboratory testing Test the membrane system for real applications Built the membrane film into a composite membrane Construct stable membrane film Produce recombinant aquaporin simulation simulation The Aquaporin (MEMBAQ) project The goal of the project is to explore the possibilities to incorporate recombinant aquaporin molecules in different types of industrial membranes for water filtration.

  5. ΔP Concept Courtesy: PH Jensen, Aquaporin Porous hydrophilic support of lipid bilayer, like mica, cellulose PorousTeflon film or other hydrophobic material Planar lipid bilayer membrane with incorporated aquaporins. Aquaporin molecule Phospholipid molecule or other amphiphilic lipid molecule

  6. MEMBAQ: 9 partners

  7. Simulations: Aid design of better aquaporins Simulations: Optimize design of nanotech membrane materials Role of Simulations in MEMBAQ At MEMPHYS, the objective is to implement computer simulations of aquaporins (AQPs) embedded in different nanotechnological membrane materials, and to use the data from computer simulations in the design of better membrane materials. Stable membrane film Testing in real applications Recombinant aquaporin

  8. .. More technical details ..

  9. SoPIP2;1: Spinach Leaf Aquaporinwill be used in MEMBAQ • Most aquaporins’ channels are always (constitutively) open • Unlike most mammalian AQPs, SoPIP2;1 is a gated channel • Sometimes open, sometimes closed • The gating is controlled by several mechanisms

  10. Crystal Structure of SoPIP2;1 • OPEN and CLOSED conformations were trapped and crystallized • OPEN and CLOSED conformations differ in certain respects Tornroth-Horsefield et al. Nature, 2006, 439, 688-694

  11. Open and Closed States of SoPIP2;1 SoPIP2;1 is a GATED water channel Open Closed Closed Open

  12. Closed Open Courtesy: Urban Johanson, Lund. U SoPIP2;1

  13. CLOSED OPEN Courtesy: Urban Johanson, Lund. U D-LOOP BLOCKING THE PORE BY LARGE MOVEMENT

  14. What drives the gating ? • Phosphorylation at Ser274 and Ser115 opens the channel • Calcium is required for keeping it closed • Protonation of His193 closes the channel All these can independently alter the conformation of the D-loop

  15. What drives the gating ? Loop D N-terminus • The D-loop links to the N-terminus via a network of H-bonds mediated by R190, D191, R118 • The network of H-bonds is broken by phosphorylation of Ser-115 Tornroth-Horsefield et al. Nature, 2006, 439, 688-694

  16. Gating by Ser115 Tornroth-Horsefield et al. Nature, 2006, 439, 688-694

  17. Overall Objectives Molecular Dynamics Simulations • Quantitative estimation of the water conduction rates through SoPIP2;1 • No experimental measurements yet • Enhance water permeation rate of SoPIP2;1 • Drive it towards a constitutively open conformation

  18. Simulation Methods and Setups

  19. (Why) Molecular Dynamics Simulations • Trajectories of molecular systems in time using Newton’s equation of motion • Time and length scales of nanoseconds and nanometers are accessible • Thermodynamic properties can be calculated

  20. - + Molecular Dynamics Simulations • Each atom represented by point mass and point charge • Interactions between atoms described by springs, electrostatics, and so on • Evolution of a molecular trajectory • Based on Newton’s classical equations of motion • Macroscopic thermodynamic properties can be calculated using the principles of statistical mechanics • No black magic !

  21. Simulation Setup Tetrameric model of SoPIP2;1 embedded in a fully hydrated (POPE) and/or phosphatidylcholine (POPC) lipid bilayer

  22. Molecular Dynamics of SoPIP2;1 in Membranes • NPT ensemble • Temperature: 310 K • Pressure: 1 atm. • N ~ 110000 atoms • 270 lipids • Protein • ~ 17000 water • ions • 105 x 105 x 80 Å • Time step: 1 x 10-15 s • CHARMM force field • Ser115 and Ser274 not phosphorylated 18 Å

  23. Simulations Implemented • CLOSED and OPEN conformations • In POPC or POPE lipid membranes • CLOSED mutants to improve Permeability • With POPC membranes

  24. Simulations Completed~ 0.3 μs, 100,000 cpu hours Simulations run on DCSC

  25. ResultsSingle Channel Permeability

  26. Single Channel Permeability jW = pf ΔCS • In principle, experimental measurements are possible • Estimation of single channel osmotic permeability is possible from equilibrium MD simulations • Estimates from simulations are usually within an order of magnitude of experimental measurements • However, RELATIVE estimates (permeability of one channel versus another) are reliable Jensen & Mouritsen (2006)Biophys. J.,90, 2270-84

  27. \]’ Single-channel Permeability Constants Osmotic Permeability (pf) www.ks.uiuc.edu Zhu, et al. (2004) Phys. Rev. Lett.,93(22), 224501

  28. Single-channel Permeability Constants Diffusive Permeability (pd) www.ks.uiuc.edu k0 = # water molecules that traverse the channel per unit time vw = Molar volume of water Zhu, et al. Phys. Rev. Lett.,93(22), 224501

  29. Single Channel Permeability Khandelia and Mouritsen, unpublished data

  30. Single Channel Osmotic Permeability pf AQP1: 6 x 10-14 cm3/s Khandelia and Mouritsen, unpublished data

  31. Low pf of SoPIP2;1 • Simulations predict an absolute pfone order of magnitude lower than AQP1, two to threefold lower than GlpF. • However, no experimental data is available for SoPIP2;1 to compare absolute values with • Ratio of pf(closed)/pf(open) is similar to experimentally measured values Suga and Maeshima (2004): Plant Cell Physiol,45(7), 823-30

  32. ResultsInfluence of Lipid Type

  33. Influence of Lipid Type The type of lipid (POPC vs. POPE) should not influence permeability

  34. ResultsEnhancing Permeability of SoPIP2;1Shifting Conformational Equilibrium towards the OPEN state In collaboration with Prof. Per Kjellbom & Dr. Urban Johanson (Lund University, Sweden)

  35. Gating of SoPIP2;1How to improve water conductivity ? • Unlike most mammalian AQPs, SoPIP2;1 and other plant aquaporins are gated • SoPIP2;1 can switch between CLOSED and OPEN states • From the MEMBAQ perspective, it is important that the conformational equilibrium of SoPIP2;1 is driven towards the OPEN state for maximal filtration efficiency • How ?

  36. Enhancing Water Conductivity 1. Role of R190 and D191 in Gating Loop D N-terminus Tornroth-Horsefield et al (2006). Nature, 439, 688-694 Arg190 and Asp191 on the gating loop anchor the loop to the Calcium ion It has been shown that homologous mutants in Arabidopsis thaliana PIP2;2 could not be closed C Tournaire-Roux, et al.(2003) Nature,425(6956), 393-7

  37. Enhancing Water Conductivity2. Longer D-loop in SoPIP2;1 Hedfalk et al. (2006) Curr. Opin. Struct. Biol, 16, 447–456

  38. Two More (Successful) Strategies Tested in Simulations • R190A-D191A mutation: Might disrupt the H-bonded network, and release the D-loop from the N-terminus • Truncation of the D-loop: Deletion of the extra residues 193 through 196 should remove steric hindrance to water transport (Both mutants of the CLOSED conformation)

  39. Simulations Completed

  40. Both Mutants Increase Permeability Khandelia and Mouritsen, unpublished data

  41. Molecular Basis for Increased Permeability

  42. Molecular Basis for Increased Permeability: Role of Ser36 Wild Type Mutant R190A-D191A Only one monomer is shown, ~ 40 ns Ser36 is conserved in PIPs

  43. Wild Type Final Initial R190-D191A

  44. Molecular Basis for Increased Permeability: Role of Ser36 Khandelia and Mouritsen, unpublished data

  45. CLOSED OPEN Courtesy: Urban Johanson, Lund. U D-LOOP BLOCKING THE PORE BY LARGE MOVEMENT

  46. Summary • Single-channel osmotic permeability constants computed • Mutants with higher water conductivity were designed, which may be more suitable for future prototypes in MEMBAQ • New fundamental insights into the molecular basis for gating of SoPIP2;1: Ser36 involved in gating ? • Mutants are being tested in the lab.

  47. Future Research • Mechanical properties of free standing and solid-supported lipid bilayers (with and without protein) • Effect of a pressure gradient on permeation dynamics, and on supported bilayer properties ΔP Courtesy: Peter Holme Jensen, Aquaporin

  48. Acknowledgements • Ole G. Mouritsen, the director of MEMPHYS • MEMBAQ, for the funding • DCSC, for the supercomputing resources. • Urban Johanson and Per Kjellbom (collaborators at LUND, Sweden)

  49. Molecular Basis for Increased Permeability: Role of L263, V264

  50. Molecular Basis for Increased Permeability: Summary

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