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Prostaglandins, Leukotrienes and Essential Fatty Acids 79 (2008) 131–134

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Prostaglandins, Leukotrienes andEssential Fatty Acids

0952-32

doi:10.1

� Corr

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journal homepage: www.elsevier.com/locate/plefa

Insights from biophysical studies on the role of polyunsaturated fatty acidsfor function of G-protein coupled membrane receptors

Klaus Gawrisch a,�, Olivier Soubias a, Mihaela Mihailescu b

a Section of NMR, Laboratory of Membrane Biochemistry and Biophysics, NIAAA, NIH, Bethesda, MD 20892, USAb Department of Physiology and Biophysics, University of California, Irvine, CA, USA

a r t i c l e i n f o

Keywords:

Docosahexaenoic acid

G-protein-coupled membrane receptor

Rhodopsin

Nuclear Magnetic Resonance

Neutron Scattering

78/$ - see front matter & 2008 Elsevier Ltd. A

016/j.plefa.2008.09.002

esponding author.

ail address: klausg@mail.nih.gov (K. Gawrisch

a b s t r a c t

The composition of the lipid matrix is critical for function of membrane proteins. Perhaps one of the

best studied examples is the function of the G-protein-coupled membrane receptor (GPCR) rhodopsin

which is located in membranes with high content of phospholipids with polyunsaturated docosahex-

aenoic acid chains (DHA, 22:6n-3). Technological advances enabled a more detailed study of structure

and dynamics of DHA chains and their interaction with rhodopsin. It was established that

polyunsaturated DHA differs from saturated and monounsaturated hydrocarbon chains by far more

rapid structural conversions. Furthermore, DHA chains tend to have higher density near the lipid/water

interface while density of saturated chains is higher in the bilayer center. The interface of rhodopsin has

a small number of sites for tighter interaction with DHA. Polyunsaturated phosphatidylethanolamines

accumulate preferentially near the protein. Surprisingly, the high conformational freedom of most DHA

chains is not measurably reduced upon interaction with rhodopsin. While some observations point at an

involvement of continuum elastic properties of membranes in modulation of rhodopsin function, there

is growing evidence for a role of weakly specific DHA–rhodopsin interactions.

& 2008 Elsevier Ltd. All rights reserved.

1. Conformation and dynamics of DHA

Ever since it was established that the disks of rod outer segmentsof the retina contain the highly unsaturated docosahexaenoic acid(DHA, 22:6n-3) at concentrations up to 50 mol% of all fatty acids,speculation arose that peculiarities in DHA structure and dynamicsmay impart unique biophysical properties on membranes that couldmodulate rhodopsin function. This was confirmed by reconstitutionof rhodopsin into bilayers with varying degree of unsaturationfollowed by a measurement of the metarhodopsin I/metarhodopsinII (MI/MII) ratio after photoactivation [1–3].

How do membranes that are rich in DHA differ from lessunsaturated ones? For a long time it is known that lipids inpolyunsaturated membranes have low order [4–7]. Initially it wasproposed that low order may result from perturbations caused byrigidity and bulkiness of polyunsaturated hydrocarbon chains.Indeed, DHA has a unique chemical structure with six cis-lockeddouble bounds. The number of degrees of freedom of DHA chainsis substantially lower which could be indicative for rigidity.Polyunsaturated chains in crystals form highly ordered, elongatedstructures with angle-iron or helical arrangement of double bonds[8]. Such structures were also observed by molecular mechanicscalculations conducted at low temperature [9,10].

ll rights reserved.

).

But in the past decade our view of DHA has changed. Earlymolecular simulations by Rabinovitch and Ripatty [11] suggestedthat the unique electronic structure of polyunsaturated chainsmay permit rapid conformational changes. Recent experimentalstudies at our laboratory as well as quantum chemical calcula-tions and molecular dynamics simulations by our collaboratorsrevealed the full magnitude of conformational flexibility of DHA[12,13]. High conformational flexibility of DHA chains wasreported by Brown and collaborators [14,15], the Davis [16], aswell as the Klein laboratory [17,18]. Our 13C MAS NMR relaxationexperiments have shown that DHA isomerizes on the timescale of1–100 ps and explore their entire conformational space within10 ns [19]. Experimental results clearly indicate that low order inbilayers with high DHA content is a direct consequence of highconformational flexibility and of rapid structural conversions ofDHA chains themselves. Quantum chemical and molecularmechanical calculations revealed that this flexibility is causedby extremely low potential barriers for changes of dihedral bondangles in vinyl bonds [12]. Low potential barriers permit thepolyunsaturated chains to rapidly change conformation withoutsignificant energetic penalty (Fig. 1).

2. Nonspecific DHA–protein interactions

In continuum models of lipid–protein interaction, the influenceof the lipid matrix on protein function can be expressed as lateral

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pressure that acts on membrane imbedded proteins. It is assumedthat the photoisomers of rhodopsin have different shape. When anintegral membrane protein changes shape, it performs workagainst the lateral pressure. As a result, the photointermediates ofrhodopsin have free energies that not only reflect the internalenergy of the protein but also the work against lateral pressurefrom the lipid matrix [20] (Fig. 2).

Lateral pressure varies along the bilayer normal. Pressures arehigher at the lipid–water interface where lipids tend to havehigher order. Differences in the pressure profile between DHA-containing bilayers and their less unsaturated counterparts couldexplain the unique influence of DHA on rhodopsin function.Unfortunately, pressure profiles cannot be measured directly. Butchanges of order parameters of lipid hydrocarbon chains mayserve as qualitative indicators for changes in the pressure profileinduced by DHA [21]. Also the distribution of hydrocarbon chain

Fig. 1. Snapshots of an 18:0–22:6n-3-PC molecule in a lipid bilayer taken from a

500 ps-window of a 10 ns-molecular simulation by Feller et al. [12]. The

polyunsaturated chain is shown in purple, the saturated chain in yellow and the

phosphocholine headgroup and glycerol region of the lipid in blue. The images

demonstrate the rapid structural conversions of DHA chains on the subnanosecond

timescale. The tendency of DHA to have higher density near the lipid water

interface is visible as well.

lateral pr

Fig. 2. Membrane proteins sense the pressure profile of a lipid bilayer. Lipids are compr

a conformational change it performs work against membrane lateral pressure which is

densities along the bilayer normal has been linked to peculiaritiesin the pressure profile [13,22].

The distribution of saturated stearic acid (18:0) andpolyunsaturated DHA in the mixed chain phosphatidylcholine18:0–22:6n-3-PC was measured by neutron scattering [23]. Lipidswere synthesized with either perdeuterated sn-1 stearic acidchains (18:0d35), or perdeuterated DHA chains (22:6n-3d11) orboth protonated stearic acid and DHA chains. Data were acquiredon the advanced neutron diffractometer/reflectometer at the NISTCenter for Neutron Research, Gaithersburg, MD. Due to the bigdifferences in scattering length densities for neutrons betweenprotonated and deuterated hydrocarbon chains, it is straightfor-ward to calculate scattering length densities of stearic acid andDHA chains over the hydrophobic core of bilayers (see Fig. 3) [23].It was observed that DHA segments are located with higherprobability near the lipid/water interface, while the segments ofsaturated chains locate with higher probability in the bilayercenter.

The results of the neutron scattering experiments corroboratedearlier conclusions about an uneven distribution of polyunsatu-rated chain density from 2H NMR experiments [13]. The DHA

essure

essed laterally by the hydrophobic effect (red arrows). When the protein undergoes

a contribution to free energy of protein conformers.

Fig. 3. Scattering length density profiles of stearic acid, DHA and water in fluid

18:0–22:6n-3-PC bilayers determined by neutron scattering measured at two

levels of relative humidity (86% r.h. and 66% r.h.). It is visible that DHA chain

density tends to be higher near the lipid/water interface while stearic acid density

is higher in the bilayer center. Water forms continuous layers outside the

hydrophobic core of lipid bilayers. DHA scattering length density is generally lower

than stearic acid density because of the lower content of deuterons in DHA (see

[23] for details).

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distribution causes lower order parameters near the methylterminal end of the saturated hydrocarbon chains with whichthey are paired in 18:0–22:6n-3-PC [6,13,15,24]. Lower orderstems from the wider space that saturated chains occupy in thebilayer center.

The uneven distribution of hydrocarbon chains, the low orderof DHA chains, and their rapid structural conversions result inaltered continuum properties of the lipid matrix. Bilayer thicknessof mixed chain, DHA-containing bilayers is similar to thethickness of saturated and monounsaturated lipid species with16 to 18 carbon atoms per chain [13,25]. Consistent with thesmaller bilayer thickness, lateral area per molecule of lipids withDHA chains is larger. The increased area could be a consequence ofthe weakly polar nature of the double bonds of DHA. This mayexplain why polyunsaturated chains prefer to locate somewhatcloser to the lipid/water interface. It may also explain the higherpermeability of polyunsaturated bilayers for water that wereported earlier [26].

The lateral pressure profile of 18:0–22:6n-3-PC bilayers wascalculated by molecular simulations [27,28]. It was observed thatthe shift of DHA chain density to the lipid/water interface iscorrelated with the migration of lateral stresses within the bilayerwhich in turn may explain the shifts in the MI/MII equilibrium[27]. Another indicator for migration of lateral stresses isformation of nonlamellar lipid phases. We reported earlier thatDHA chains in phosphatidylethanolamines facilitate formation ofinverted hexagonal phases [29]. Lipid monolayers of phosphati-dylethanolamines have a smaller lateral area in the headgroupregion and lager area in the region of hydrocarbon chains. Themonolayers are under negative curvature elastic stress whenforced into a bilayer arrangement. Levels of stress in 18:0–22:6n-3-PE membranes are similar to 18:1n-9–18:1n-9-PE, the goldenstandard for lipids with negative curvature stress [29]. Brown andcolleagues reported that 18:1n-9–18:1n-9-PE in bilayers favorsformation of the G-protein activation competent MII photointer-mediate of rhodopsin [30]. Since the 18:0–22:6n-3-PE is a verycommon lipid, in particular in neuronal membranes, it is reason-able to assume that those membranes are under considerablenegative curvature elastic stress as well.

Fig. 4. Sites of rhodopsin in a 18:0–22:n-3PC/PE/cholesterol bilayer that have been

proposed to interact preferentially with DHA (blue) stearic acid (STEA, red) or

cholesterol (CHOL, purple). Sites of preferential interaction were identified in a

molecular dynamics simulation by Grossfield et al. [37] (copyright (2006) National

Academy of Sciences, U.S.A.).

3. Specific DHA–protein interactions

An alternative hypothesis to explain the shifts in rhodopsinfunction is to assume that the free energy of rhodopsin is underinfluence from weakly specific interactions with DHA, similar toligand–protein interactions. In recent years, several examples ofspecific interactions between proteins and polyunsaturated chainswere reported, e.g. the interaction of arachidonic acid withprostaglandin synthase [31], the interaction of DHA with thebinding pocket of human fatty acid binding protein [32], theinteraction of the arachidonyl inhibitor with the binding pocket offatty acid amide hydrolase [33], the interaction of fatty acids withthe retinoic acid-related orphan receptor ß [34], and last but notleast, the interaction of retinal with rhodopsin [35].

In all instances, binding of polyunsaturated chains to proteinswas stabilized by major contributions from polar, noncovalentinteractions like salt bridges, direct hydrogen bonding betweenhydroxyl or carbonyl groups to amino acids, or indirect hydrogenbonding via water molecules. But those interactions do notexplain selectivity to the number and location of double boundsin the chains. The attractive p�p interactions that may occurbetween double bonds of polyunsaturated fatty acids andaromatic side chains of amino acids may be the missing link.Indeed, it is remarkable that the fatty acid binding pockets in theproteins that are listed above are lined by a very high density of

the aromatic amino acids phenylalanine, tyrosine, and trypto-phan.

Recent NMR results obtained at our lab provide some evidencefor involvement of specific interactions with DHA in rhodopsinfunction. In experiments on rhodopsin that was reconstituted intomembranes with variable content of phosphatidylcholine, phos-phatidylethanolamine, and phosphatidylserine, all with DHAhydrocarbon chains, it was observed that DHA chains competefor contact with a small number of weakly specific sites onrhodopsin [36]. While specificity was caused by DHA, the lipidheadgroups modulated interaction strength. Interactions werestrongest for PE, followed by PS, and PC [36]. Monounsaturatedlipid species have preferential contact with a different site onrhodopsin. The lifetimes of associations between lipids andrhodopsin are short, indicating that mutual attraction has modeststrength. According to our NMR measurements, all lipids thatsurround the protein in a first layer are replaced withinmicroseconds or even shorter times.

The conclusion is that rhodopsin has sites for preferentialinteraction with particular lipid species. Molecular simulationssuggest as well that the surface of rhodopsin is inhomogeneous interms of lipid–rhodopsin interaction (see Fig. 4) [37]. DoesDHA–rhodopsin interaction depend on the state of photoactiva-tion of rhodopsin? To answer this question, rhodopsin wasbleached during the NMR studies. Experiments were conductedat high and low pH to yield the photointermediates MI or MII,respectively. Neither of those two photointermediates showeddifferences in the interaction with DHA compared to dark adaptedrhodopsin. But the photointermediate MIII had stronger contactswith DHA [36]. Interaction of opsin with DHA was similar to darkadapted rhodopsin.

Specificity in lipid–rhodopsin interaction may shift lipidcomposition in the protein annulus compared to the bulk of thelipid matrix. Lipids that have higher affinity to rhodopsin will beenriched near the protein, despite the rapid exchange of lipids.Indeed, by NMR saturation transfer from protein to lipids, weobtained evidence that polyunsaturated phosphatidylethanola-mines are partially enriched near rhodopsin [36].

How may specific interactions between lipids and proteininfluence protein function? The double bonds of polyunsaturatedchains could be attracted to aromatic amino acids of GPCR thatstabilize a particular conformation of the protein. Polyunsaturatedhydrocarbon chains may interfere with interactions inside theGPCR if the chains could reach into the protein. There is evidencefor such events from molecular simulations [38]. Interaction with

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phospholipids may also interfere with hydrogen bonding and saltbridges that form between transmembrane helices, as well aswith the ubiquitous van der Waals interactions.

4. Summary

Recent NMR studies have improved our understanding of therole of polyunsaturated fatty acids like DHA in lipid bilayers. TheDHA chains are special because they interconvert betweenconformations more rapidly, they prefer to locate in the hydro-phobic core of bilayers near the lipid/water interface, and they arecapable of engaging in partially specific interactions with GPCRlike rhodopsin. Bilayers rich in DHA may alter protein functionboth by a change of general membrane properties as well as byspecific interactions with particular regions of the protein.Experiments that are currently underway at our laboratorysuggest that GPCR adjust their structure far more nimbly to thelipid environment than generally assumed. It is not just the lipidmatrix that deforms in response to the needs of the protein, butthe protein may adjust structurally to the lipid matrix as well [39].The latter aspect deserves more attention in future investigations.

Acknowledgements

This work was supported by the Intramural Research Programof NIAAA, NIH. The neutron diffraction studies were conducted onthe AND/R instrument, constructed by the Cold Neutrons forBiology and Technology (CNBT) partnership, supported by theNational Institute of Standards and Technology, the Regents of theUniversity of California, and by a grant RR14812 from the NationalInstitute for Research Resources awarded to the University ofCalifornia at Irvine.

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