Interactions of lipid membranes and small moleculesA thermodynamic approach Peter Westh NSM, Biomolecules Roskilde Univ. firstname.lastname@example.org
A matter of polarity ! Synopsis • Membrane embedded solutes (fatty acids and alkanes) • Partitioned solutes (alcohols, amino acids, sulfoxides…) • Hydrophilic (aqueous) solutes (glycerol, sugars, polyalcohols…)
One crucial nanometerPerturbations by ”foreign compounds” • Polar compounds • Osmolytes • Salts • Neurotransmitters Interface ~1-1.5 nm • Amphiphiles • Alcohols • sulfoxides • Hydrophobes • Fatty acids • alkanes
Non-polar solutes Fatty acids
Free fatty acids in membranesIn vitro • High Kp For C18OOH, for example, Kp~107. Hence for at typical lab-sample (0.1% lipid in aqueous solution), 99.99% of the added fatty acid will be partitioned. For small FA (e.g. C10OOH) it is only ~70% Høyrup et al J.Phys.Chem.B (2001) 105; 2649
Free fatty acids in membranesIn vivo • The accumulation in membranes is much less pronounced due competition with binding to e.g. serum albumin. • Typically 0.3-10%(w/w) – types strongly influenced by the diet Kp~107 KB~107 • Free fatty acids in biological membranes effect a number of processes: Intermembrane cholesterol traficking Transmembrane metabolic energy flow Drug partitioning and uptake Permeability Activation/inhibition of membrane proteins Inter- and intra cellular signaling Lipid ”raft” segregation Cryo- susceptibility Etc etc.
Membrane-fatty acid complexes – Structure Snapshots – DMPC/OA Snapshots – DMPC/SA MD simulation, Peters et al., in prep • 32 FFA XFFA = 0.2 • 128 DMPC • ~5000-6500 water • Counter ions • 40-60 ns • NPT ensemble
Phase behaviorDMPC-SA and DMPC OA DMPC-SA: DSC measurements Ortiz & Fernandez (1987) Chem Phys Lip45; 75.
Fatty acids in DMPC Densitometry- molecular packing OA SA The fatty acid-membrane complex is more loosely packed than pure DMPC The perturbing effect (per molecule of fatty acid) is particularly strong at low xFA. DMPC-SA phase boundary Apparent volume af fatty acid in DMPC @ 40C ~536 Å3. The MD simulation yields 520-540Å3. Pure acids ~536 Å3 Peters et al., in prep
Order parameters Where Q is the angle between the C-D and the bilayer normal Quantifies the balance of trans gauche conformers (0-1) SCD~1 SCD< 1 At xFA=0.20: PROTONATED fatty acids order the acyl chains of the lipid ANIONIC fatty acids don’t (or very little so) A SATURATED fatty acid orders the acyl chains more than an UNSATURATED Peters et al., in prep
Effects of fatty acids depend strongly on the H-FA H+ +FA- equilibrium Peters et al., in prep
(Exagerated) picture of protonation effects Fatty acid anion. Surface area per FA: OA-~40Å2, SA-~29Å2 Note that: pKa for free fatty acids in membranes is ~ 6.5-7.5 (it is ~5 in water). It follows that the change between these two pictures readily occurs under physiological conditions. Many biophysical results are reported without specifying pH!! Protonated fatty acid. Surface area per FA: HOA~17Å2, HSA~7Å2 Andersen et al. (2007) Roskilde Univ. Library.
End-to-end distances of fatty acids in DMPC Fully stretched SA: 21.2 Å Kinked conformations are common to OA – not to SA. Peters et al., in prep
Amphiphilic solutes 1-alkanols (normal alcohols)
Lipid membranes and solutes of intermediate polarity Menbrane partitioning A (aq) A(mem) Kp=[A (mem)] / [A (aq)] Thermodynamic approach DG=-RTlnKp In principle one then differentiates with respect to T,P and ni to obtain other thermodynamic functions for the partitioning process (e.g. DH, DCp DV etc.)
Bulk Partitioning The distribution between two phases If A is dissolvedin the two phases we may approximate a chemical potential At equilibrium Equilibrate The dependence on the environtmental parameters n,P,T gives other thermodynamc functions Separate Analyze
Membrane partitioning Equilibrate Membrane partitioning coefficients are difficult to measure (the separation step is generally impossible) Moreover The classification [of ligand] into dissolved and bound molecules is an extra-thermodynamic and somewhat arbitrary procedure. Terrell Hill 1963. Separate Analyze In addition: Non-ideality (anisotropy, size difference) ”real” concentration (water penetration) Pedersen et al (2007) Biophys. Chem.125, 104.
Small alcohols and DMPC Titration calorimetry Partitioning is concentration independent – Surface adsorbtion may saturate in a Langmuir style MAS-NMR Holte and Gawrich (1997) Biochem36, 4669. Trandum et al (1999) BBA. 1440; 179.
Partitioning and affinity Manometer Water+alcohol+ liposomes Water+alcohol Cell Reference Water+alcohol Water+alcohol+ liposomes Water Water+alcohol Water+alcohol+ liposomes It the alcohol-membrane interaction attractive or repulsive? Westh et al. (2001) Biophys. Chem.89: 53.
Thermodynamics vs. partitioning Thermodynamic binding parameter Structural (”Kp”) binding parameter Partitioning provides a realistic picture for 1-butanol and more hydrophobic alcohols
Binding and occupancy High affinity (K>>1): Bindingoccupancy (G=L) Low affinity (K~1): Occupancy > binding (L>G) ”Low affinity” requires a different molecular picture. E.g. In stead of
Co-partitioning of water Methanol and POPC: Lipid in green, methanol in blue and selected water molecules in red. ” Patra et al (2004) Condensed Matter
General validity of the partitioning scheme • For solutes more hydrophobic than 1-propanol it provides an effective and very simple framework to discuss membrane-solute interactions. • For less hydrophobic solutes it becomes gradually less useful and for physiologically important solutes such as salts, small saccharides (e.g. glucose) and polyhydroxy alcohols (e.g. sorbitol) it is of little value. This implies that water interacts more favorable with the membrane than the solute does.
Membrane-(1-)alkanol interactions • Enormous literature available Interesting probe for general relationships of membrane perturbations • Biological relevance Anesthesia/intoxication Otherwise limited
1-hexanol in DMPC 40 nsec MD simulation Pedersen et al (2007) Biophys. Chem.125, 104.
Membrane-alcohol complexesStructure Ethanol-DPPC Octanol-DPPC Holte and Gawrich (1997) Biochem36, 4669.
Interchelated and interfacial positions Dodecanol Octanol Lund (2007) Roskilde Univ. Library Patra et al (2004) Condensed Matter Similar results have been found by NMR spectroscopy. Thewalt & Cushley (1987) BBA905, 329. Pope et al (1984) Chem Phys Lip35, 259
Alcohol permeability Z-position os all alcohol molecules in DPPC. Ethanol (red) methanol (green). Crossing events frequent for EtOH – never seen for MeOH (within 40 ns) N~3-4 Patra et al (2004) Condensed Matter Permeability coefficient for Fickean permeation: Brahm (1983) J. Gen Physiol81, 283.
Molecular packing of alcohols in DMPC DV=Vapp-V (standard either pure alcohol or dilute aqueous solution) Aagaard et al (2005) Biophys. Chem. 119; 61
Simulation of DMPC-hexanol • Effects og hexanol: • Slight ordering C1-C7 • Large free volume (and disordering) for C8-C12. • DVteoretical=DVexp=4 cm3/g • Vfree~14 cm3/g Pedersen et al 2007
Thickness and lateral mobilityAlcohol-lipid membrane DoOH, 40C OcOH, 40C OcOH, 30C HexOH, 30C HexOH, 50C MD simulation, DMPC-HxOH Pedersen et al (2007) Biophys. Chem.125, 104. SANS data, Unilamellar DMPC Lund (2007) Roskilde Univ. Library Strongly mismatched alcohols – e.g. 1-Hexanol – makes the membrane thinner and more ”laterally dynamic” Matched 1-alkohols makes it thicker, and more ordered and dense.
Polar solutes Trehalose and other small sugars
Highly polar solutes – do not partition but exert pronounced effects on membranes • The key is the distribution in the interfacial layer Koynova et al (1987) Eur. Biophys. J.,25:261. Water has higher affinity for interface than solute Preferential hydration Solute has higher affinity for interface than water Preferential binding Preferential binding favors large interfacial areas (and vice versa)
Trehalose: a chemical chaperone • This disaccharide has remarkable stabilizing effects both in vitro and in vivo • E.g. • Accumulated (2-20%w/w) in extremely drought tolerant animals • Retains integrity of freeze-dried liposomes • Etc etc What is the mechanism of the stabilization of membranes provided by trehalose (and other saccharides)? Membrane-trehalose interactions Vitrification
Applications –stabilization an more • Stabilization of vaccines • Hypothermal organ storage • Treatment of dry-eye syndrom and dry skin in humans • Cosmetics (fatty acid anti-oxidant??) • Suppression of free radical damage • Protection against anoxic damage • Inhibition of dental caries • Enhance yeast ethanol production • Stabilize flavor in foods • Protects plant material against physical stresses • Suppresses Osteoclast differentiation (anti-osteoporotic drug) • Blood platelet storage • Anti protein aggregation (drug against Huntington’s desease) • Inhibits toluene toxicity • Inhibits senescence in cut flowers Effects both during water stress (freezing, dehydration etc) and in fully hydrated systems And both in living cells and purified macromolecules and macromelecular assemblies
MD simulations: PC-trehalose attraction •  B.W. Lee, et al., (2004) Fluid Phase Equil. 225 63-68. •  B.W. Lee, et al., (2005) Fluid Phase Equil. 228 135-140. •  S. Leekumjorn, A.K. Sum, (2006) Molec. Simulation, 32 219-230. •  S. Leekumjorn, A.K. Sum, (2006) Bioph. J. 90 3951-3965. •  C.S. Pereira, P.H. Hunenberger, (2006) J. Phys. Chem. B, 110 15572-15581. •  C.S. Pereira, et al., (2004) Biophys. J. 86 2273-2285. •  A. Skibinsky, et al., (2005) Biophys. J. 89 4111-4121. •  A.K. Sum, (2005) Chem. Biodivers., 2 1503-1516. •  A.K. Sum, et al., (2003) Biophys. J. 85 2830-2844. •  M.A. Villarreal, et al., (2004) Langmuir, 20 7844-7851. Periera et al 2004 Vilareal et al 2004
Vapor pressure measurements Manometer Thermodynamic definition of binding: DP=0: ”neutral” water and trehalose interacts equally well with PC-membrane DP>0: Sugar binds stronger than water DP<0: Water binds stronger than sugar Cell Reference Water+trehalose +liposomes Water+trehalose Molecular interpretation: (for e.g.DP<0) membrane
Water binds stronger (DP<0) On the average more than a monolayer (~17 H2O) is complete devoit of trehalose
Interfacial effects account for the phase behavior Temperature scanning: The degree of preferential exclusion scales with the surface area.
Mechanism of partiel depletionSurface accessibility Solvent simulation: Jesper S. Hansen Surface assignment: Erik Tuchsen Membrane simulation: Morten Ø. Jensen Accounts for 50% of the experimentally observed depletion Other factors include favorable membrane-water and water-solute interactions