Interactions of lipid membranes and small molecules

A thermodynamic approach

Peter WesthNSM, Biomolecules

Roskilde Univ. pwesth@ruc.dk

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”

Interface ~1-1.5 nm

Polar compounds• Osmolytes• Salts• Neurotransmitters

Amphiphiles• Alcohols• sulfoxides

Hydrophobes• Fatty acids• alkanes

Non-polar solutes

Fatty acids

Free fatty acids in membranesIn vitro

• High Kp

Høyrup et al J.Phys.Chem.B (2001) 105; 2649

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%

• 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

Free fatty acids in membranesIn vivo

Kp~107 KB~107

• Free fatty acids in biological membranes effect a number of processes:

Intermembrane cholesterol trafickingTransmembrane metabolic energy flowDrug partitioning and uptakePermeabilityActivation/inhibition of membrane proteins

Inter- and intra cellular signalingLipid ”raft” segregationCryo- susceptibilityEtc etc.

Membrane-fatty acid complexes – Structure

Snapshots – DMPC/OA Snapshots – DMPC/SA

32 FFA XFFA = 0.2

128 DMPC

~5000-6500 water

Counter ions

40-60 ns

NPT ensemble

MD simulation, Peters et al., in prep

Phase behaviorDMPC-SA and DMPC OA

Ortiz & Fernandez (1987) Chem Phys Lip 45; 75.

DMPC-SA:

DSC measurements

Fatty acids in DMPC Densitometry- molecular packing

Pure acids

~536 Å3

Volume properties of Stearic acid and Oleic acid in L DMPC

40oC

xFA in membrane

0.00 0.05 0.10 0.15 0.20 0.25 0.30

Va

pp (f

att

y a

cid

)

(cm3 /g

)

1.1

1.2

1.3

1.4

VA

pp (

Å3 /m

ole

cule

)

500

550

600

650

OleicStearicStearic IIStearic 2006

Phase boundary DMPC-stearic acid

xFA

0.00 0.05 0.10 0.15 0.20 0.25 0.30

non

aqV

app

(ml/g

)

0.980

0.985

0.990

0.995

1.000

1.005 OA

SA

DMPC-SA phase boundary

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.

Apparent volume af fatty acid in DMPC @ 40C ~536 Å3.

The MD simulation yields 520-540Å3.

Peters et al., in prep

Order parameters

|2/1)(cos2/3| 2 CDS Where 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

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!!

Andersen et al. (2007) Roskilde Univ. Library.

Fatty acid anion.Surface area per FA: OA-~40Å2, SA-~29Å2

Protonated fatty acid.Surface area per FA: HOA~17Å2, HSA~7Å2

End-to-end distances of fatty acids in DMPC

protonated OA in DMPC (T=330K)protonated OA in DMPC (T=330K)

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 approachG=-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

aqx

aqorgx

org axax ][ ][

The distribution between two phases

Separate

Analyze

Equilibrate

0

2

0

1H

TTT

G

p

If A is dissolved in the two phases we may approximate a chemical potential

At equilibrium

The dependence on the environtmental parameters n,P,T gives other thermodynamc functions

Membrane partitioningMembrane 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.

Pedersen et al (2007) Biophys. Chem. 125, 104.

In addition:

Non-ideality (anisotropy, size difference)

”real” concentration (water penetration)

Separate

Analyze

Equilibrate

Small alcohols and DMPC

+ + + - -

= 0

= 0 = 0

Holte and Gawrich (1997) Biochem 36, 4669. Trandum et al (1999) BBA. 1440; 179.

MAS-NMR

Partitioning is concentration independent – Surface adsorbtion may saturate in a Langmuir style

Titration calorimetry

Partitioning and affinity

Methanol

0.0 0.2 0.4 0.6 0.8

0

1

2

3

Ethanol

0.0 0.2 0.4 0.6 0.8 1.0

P

(m

bar

)

0

1

2

3

4

1-Propanol

m3 (mol /kg water)

0.0 0.1 0.2 0.3 0.4

0

1

2

1-Butanol

0.0 0.1 0.2 0.3 0.4

0

1

2

1-Pentanol

0.00 0.05 0.10 0.15

P

(m

bar

)

0.0

0.5

1.0

1.5

1-Hexanol

m3 (mol/kg water)

0.00 0.02 0.04 0.06 0.08

0.0

0.2

0.4

0.6Water+alcohol

+liposomes

Water

Manometer

Cell Reference

It the alcohol-membrane interaction attractive or repulsive?

Westh et al. (2001) Biophys. Chem. 89: 53.

Water+alcohol

Water+alcohol

Water+alcohol+liposomes

Water+alcohol+liposomes

Water+alcohol

Thermodynamics vs. partitioning

APTlip

AlipA n

n

,,

lip

lipA

lipA dn

dn

Thermodynamic binding parameter

Structural (”Kp”) binding parameter

Partitioning provides a realistic picture for 1-butanol and more hydrophobic alcohols

Binding and occupancy

00 22 HAMAHM

00 22 HAMAHM

High affinity (K>>1):

Bindingoccupancy (

Low affinity (K~1):

Occupancy > binding

”Low affinity” requires a different molecular picture.

E.g. In stead of AMAM

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 relevanceAnesthesia/intoxicationOtherwise limited

1-hexanol in DMPC

40 nsec MD simulation

Pedersen et al (2007) Biophys. Chem. 125, 104.

Membrane-alcohol complexesStructure

+ + + - -

= 0

= 0 = 0

Holte and Gawrich (1997) Biochem 36, 4669.

Ethanol-DPPCOctanol-DPPC

Interchelated and interfacial positions

Similar results have been found by NMR spectroscopy.

Thewalt & Cushley (1987) BBA 905, 329.Pope et al (1984) Chem Phys Lip 35, 259

Dodecanol

Octanol

Lund (2007) Roskilde Univ. LibraryPatra et al (2004) Condensed Matter

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)

Permeability coefficient for Fickean permeation:

x

DKP p

N~3-4

Patra et al (2004) Condensed Matter

Brahm (1983) J. Gen Physiol 81, 283.

Molecular packing of alcohols in DMPCV=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:

1. Slight ordering C1-C72. Large free volume (and disordering)

for C8-C12.3. Vteoretical=Vexp=4 cm3/g4. Vfree~14 cm3/g

Pedersen et al 2007

Thickness and lateral mobilityAlcohol-lipid membrane

HexOH, 50C

HexOH, 30C

OcOH, 30C

OcOH, 40C

DoOH, 40C

SANS data, Unilamellar DMPCLund (2007) Roskilde Univ. Library

MD simulation, DMPC-HxOHPedersen et al (2007) Biophys. Chem. 125, 104.

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

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)

Koynova et al (1987) Eur. Biophys. J., 25:261.

Trehalose: a chemical chaperone

This disaccharide has remarkable stabilizing effects both in vitro and in vivoE.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

• [1] B.W. Lee, et al., (2004) Fluid Phase Equil. 225 63-68.

• [2] B.W. Lee, et al., (2005) Fluid Phase Equil. 228 135-140.

• [3] S. Leekumjorn, A.K. Sum, (2006) Molec. Simulation, 32 219-230.

• [4] S. Leekumjorn, A.K. Sum, (2006) Bioph. J. 90 3951-3965.

• [5] C.S. Pereira, P.H. Hunenberger, (2006) J. Phys. Chem. B, 110 15572-15581.

• [6] C.S. Pereira, et al., (2004) Biophys. J. 86 2273-2285.

• [7] A. Skibinsky, et al., (2005) Biophys. J. 89 4111-4121.

• [8] A.K. Sum, (2005) Chem. Biodivers., 2 1503-1516.

• [9] A.K. Sum, et al., (2003) Biophys. J. 85 2830-2844.

• [10] M.A. Villarreal, et al., (2004) Langmuir, 20 7844-7851.

Periera et al 2004

Vilareal et al 2004

Vapor pressure measurements

Water+trehalose +liposomes

Water+trehalose

ManometerThermodynamic definition of binding:

P=0: ”neutral”water and trehalose interacts equally well with PC-membrane

P>0: Sugar binds stronger than water

P<0: Water binds stronger than sugar

Molecular interpretation:(for e.g. P<0)

membrane

Cell Reference

Water binds stronger (P<0)Thermodynamic binding parameter

mol trehalose (kg Water)-1

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

(dm

treh

a/dm

DM

PC)

(

mol

/mol

)

-1.0

-0.8

-0.6

-0.4

-0.2

0.0Ser 2Ser 3Ser 4Ser 5Plot 1 Regr

P vs. mtrehalose

mol trehalose (kg water)-1

0.0 0.5 1.0 1.5 2.0 2.5 3.0

P (

Bar

)

-1000

-800

-600

-400

-200

0

On the average more than a monolayer (~17 H2O) is complete devoit of trehalose

Interfacial effects account for the phase behavior

T (oC)

23 24 25 26 27 28 29

P (

bar

)

-125

-100

-75

1

-0.45

-0.40

-0.35P

1Temperature scanning: The degree of preferential exclusion scales with the surface area.

Mechanism of partiel depletion

Surface accessibility

Membrane simulation:Morten Ø. Jensen

Surface assignment:Erik Tuchsen

Solvent simulation:Jesper S. Hansen

Accounts for 50% of the experimentally observed depletion

Other factors include favorable membrane-water and water-solute interactions