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)
Revealing the Functional Role of Reentrant Loops
Single Glycerol per channel
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.
Steered Molecular Dynamics constant force (250 pN) constant velocity (30 Å/ns)
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
Constructing the Potential of Mean Force 4 trajectories v = 0.03, 0.015 Å/ps k = 150 pN/Å
Constructing the Potential of Mean Force – cont. zp zc determined by minimizing the difference between (d) and (e)
Features of the Potential of Mean Force Captures major features of the channel The largest barrier 7.3 kcal/mol; exp.: 9.61.5 kcal/mol
Asymmetry of the Potential of Mean Force phosphorylation Cytoplasm Periplasm Asymmetric Profile in the Vestibules
Assymetric structure; biological implication? OmpF Maltoporin GlpF AQP1
Asymmetric structure of maltoporin
SMD Simulation of Glycerol Passage Trajectory of glycerol pulled by constant force
Aquaporins’ Mechanisms of Selectivity Protons, ions, and charged molecules cannot be transported. Aquaglyceroporins (GlpF) transport linear sugars in a highly stereo-selective manner
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
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
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
Selectivity filter
Evidence for Stereoselectivity of Glycerol H H OH H OH H H OH H OH H HO H H OH H Cannot be verified by experimental measurements.
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
Evidence for Stereoselectivity by Interactive Molecular Dynamics Ribitol Optimal hydrogen bonding and hydrophobic matching Arabitol 10 times slower
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
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
Glycerol-Free GlpF
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
Correlated Movement of Water in the Channel The single file of water molecules is maintained.
Diffusion of Water in the channel One dimensional diffusion: Experimental value for AQP1: 0.4-0.8 e-5
Water Bipolar Configuration in Aquaporins E.T. et al., Science 296, 525 (2002)
Proton transfer through water
Electrostatic Stabilization of Water Bipolar Arrangement E.T. et al., Science 296, 525 (2002)
Proton Blocking by a Global Orientation Mechanism