Evidence for Specificity in Lipid-Rhodopsin Interactions

The interaction of bovine rhodopsin with poly- and monounsaturated lipids was studied by 1H MAS NMR with magnetization transfer from rhodopsin to lipid. Experiments were conducted on bovine rod outer segment (ROS) disks and on recombinant membranes containing lipids with polyunsaturated, docosahexaenoyl (DHA) chains. Poly- and monounsaturated lipids interact specifically with different sites on the rhodopsin surface. Rates of magnetization transfer from protein to DHA are lipid headgroup-dependent and increased in the sequence PC < PS < PE. Boundary lipids are in fast exchange with the lipid matrix on a time scale of milliseconds or shorter. All rhodopsin photointermediates transferred magnetization preferentially to DHA-containing lipids, but highest rates were observed for Meta-III rhodopsin. The experiments show clearly that the surface of rhodopsin has sites for specific interaction with lipids. Current theories of lipid-protein interaction do not account for such surface heterogeneity. The interaction of bovine rhodopsin with poly- and monounsaturated lipids was studied by 1H MAS NMR with magnetization transfer from rhodopsin to lipid. Experiments were conducted on bovine rod outer segment (ROS) disks and on recombinant membranes containing lipids with polyunsaturated, docosahexaenoyl (DHA) chains. Poly- and monounsaturated lipids interact specifically with different sites on the rhodopsin surface. Rates of magnetization transfer from protein to DHA are lipid headgroup-dependent and increased in the sequence PC < PS < PE. Boundary lipids are in fast exchange with the lipid matrix on a time scale of milliseconds or shorter. All rhodopsin photointermediates transferred magnetization preferentially to DHA-containing lipids, but highest rates were observed for Meta-III rhodopsin. The experiments show clearly that the surface of rhodopsin has sites for specific interaction with lipids. Current theories of lipid-protein interaction do not account for such surface heterogeneity. Rhodopsin is the light receptor responsible for dim light vision in the rod photoreceptor cells of vertebrates. A large body of research demonstrated that efficiency of the rhodopsin-dependent steps of the visual process is exquisitely sensitive to membrane lipid composition, in particular to the content of ω-3 polyunsaturates (1Niu S.L. Mitchell D.C. Litman B.J. J. Biol. Chem. 2001; 276: 42807-42811Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 2Mitchell D.C. Niu S.L. Litman B.J. Lipids. 2003; 38: 437-443Crossref PubMed Scopus (101) Google Scholar, 3O'Brien D.F. Costa L.F. Ott R.A. Biochemistry. 1977; 16: 1295-1303Crossref PubMed Scopus (121) Google Scholar). Retinal membranes of mammals, similar to synaptosomal membranes in brain, contain up to 50 mol % of docosahexaenoic acid (DHA, 2The abbreviations used are: DHA, docosahexaenoic acid; ROS, rod outer segment; PIPES, 1,4-piperazinediethanesulfonic acid; CP, cross-polarization; ST-MAS-NMR, saturation transfer magic angle spinning NMR spectroscopy; PE, phosphatidylethanolamine; PS, phosphatidylserine; rf, radiofrequency; OG, octylglucoside; PC, phosphatidylcholine; OA, oleic acid. 2The abbreviations used are: DHA, docosahexaenoic acid; ROS, rod outer segment; PIPES, 1,4-piperazinediethanesulfonic acid; CP, cross-polarization; ST-MAS-NMR, saturation transfer magic angle spinning NMR spectroscopy; PE, phosphatidylethanolamine; PS, phosphatidylserine; rf, radiofrequency; OG, octylglucoside; PC, phosphatidylcholine; OA, oleic acid. 22:6n3), a polyunsaturated fatty acid with 22 carbon atoms and six double bonds that are evenly distributed over the length of the chain (4Salem N. Litman B. Kim H.Y. Gawrisch K. Lipids. 2001; 36: 945-959Crossref PubMed Scopus (758) Google Scholar). In earlier reconstitution experiments with bovine rhodopsin it was established that the equilibrium concentration of Meta-II rhodopsin increased with the concentration of DHA hydrocarbon chains in the lipid matrix (5Wiedmann T.S. Pates R.D. Beach J.M. Salmon A. Brown M.F. Biochemistry. 1988; 27: 6469-6474Crossref PubMed Scopus (144) Google Scholar, 6Mitchell D.C. Straume M. Litman B.J. Biochemistry. 1992; 31: 662-670Crossref PubMed Scopus (145) Google Scholar, 7Gibson N.J. Brown M.F. Biochemistry. 1993; 32: 2438-2454Crossref PubMed Scopus (163) Google Scholar). Also the headgroups of phospholipids had a significant effect on Meta-II formation (5Wiedmann T.S. Pates R.D. Beach J.M. Salmon A. Brown M.F. Biochemistry. 1988; 27: 6469-6474Crossref PubMed Scopus (144) Google Scholar, 7Gibson N.J. Brown M.F. Biochemistry. 1993; 32: 2438-2454Crossref PubMed Scopus (163) Google Scholar) and activation of Gt (8Alves I.D. Salgado G.F. Salamon Z. Brown M.F. Tollin G. Hruby V.J. Biophys. J. 2005; 88: 198-210Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 9Wang Y. Botelho A.V. Martinez G.V. Brown M.F. J. Am. Chem. Soc. 2002; 124: 7690-7701Crossref PubMed Scopus (45) Google Scholar). It was observed that phosphatidylethanolamines (PE) and the negatively charged phosphatidylserines (PS) increased the amount of Meta-II. The influence of PS on the equilibrium was assigned to changes of the membrane electric surface potential (7Gibson N.J. Brown M.F. Biochemistry. 1993; 32: 2438-2454Crossref PubMed Scopus (163) Google Scholar). In contrast, the sensitivity of membranes to PE content correlated with an alteration of membrane curvature elasticity (10Botelho A.V. Gibson N.J. Thurmond R.L. Wang Y. Brown M.F. Biochemistry. 2002; 41: 6354-6368Crossref PubMed Scopus (154) Google Scholar) as proposed for other membrane proteins by Navarro et al. (11Navarro J. Toiviokinnucan M. Racker E. Biochemistry. 1984; 23: 130-135Crossref PubMed Scopus (174) Google Scholar), Jensen and Schutzbach (12Jensen J.W. Schutzbach J.S. Biochemistry. 1984; 23: 1115-1119Crossref Scopus (73) Google Scholar), Gruner (13Gruner S.M. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3665-3669Crossref PubMed Scopus (550) Google Scholar), Lindblom and co-workers (14Andersson A.S. Rilfors L. Oradd G. Lindblom G. Biophys. J. 1998; 75: 2877-2887Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar), and Cantor (15Cantor R.S. J. Phys. Chem. B. 1997; 101: 1723-1725Crossref Scopus (299) Google Scholar). Litman and co-workers (6Mitchell D.C. Straume M. Litman B.J. Biochemistry. 1992; 31: 662-670Crossref PubMed Scopus (145) Google Scholar, 16Niu S.L. Mitchell D.C. Litman B.J. J. Biol. Chem. 2002; 277: 20139-20145Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar) found a correlation between mobility and orientation of the fluorescence probe DPH in lipid bilayers, summarized as a membrane free volume parameter, and Meta-II formation. Furthermore, they observed a preference of rhodopsin to locate in domains rich in di-22:6n3-PC that formed in di-16:0-PC/di-22:6-PC/cholesterol mixtures. Mouritsen proposed a link between hydrophobic thickness of lipid bilayers and activity of membrane proteins (18Mouritsen O.G. Bloom M. Biophys. J. 1984; 46: 141-153Abstract Full Text PDF PubMed Scopus (614) Google Scholar). Brown and co-workers (10Botelho A.V. Gibson N.J. Thurmond R.L. Wang Y. Brown M.F. Biochemistry. 2002; 41: 6354-6368Crossref PubMed Scopus (154) Google Scholar) suggested that Meta-II has a greater hydrophobic thickness than Meta-I, and that the lipid bilayer has to thicken to match the hydrophobic length of Meta-II. They combined hydrophobic mismatch and curvature elasticity into a flexible surface model that links membrane elastic properties to rhodopsin function in a very general way (10Botelho A.V. Gibson N.J. Thurmond R.L. Wang Y. Brown M.F. Biochemistry. 2002; 41: 6354-6368Crossref PubMed Scopus (154) Google Scholar). In this project we explored whether the rhodopsin surface should be viewed as homogeneous and the surrounding membrane as a continuum, or if specific interactions with lipids could play a role in rhodopsin activation. Lipids could associate with particular sites on the protein and alter function, in analogy to lipid-like substances that act as ligands. Such specific interactions are likely to depend on the chemical composition of lipid hydrocarbon chains, in particular polyunsaturation, and on lipid headgroups. Indeed crystal structures of bacteriorhodopsin (19Grigorieff N. Ceska T.A. Downing K.H. Baldwin J.M. Henderson R. J. Mol. Biol. 1996; 259: 393-421Crossref PubMed Scopus (870) Google Scholar, 20Mitsuoka K. Hirai T. Murata K. Miyazawa A. Kidera A. Kimura Y. Fujiyoshi Y. J. Mol. Biol. 1999; 286: 861-882Crossref PubMed Scopus (236) Google Scholar, 21Belrhali H. Nollert P. Royant A. Menzel C. Rosenbusch J.P. Landau E.M. Pebay-Peyroula E. Structure. 1999; 7: 909-917Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar, 22Luecke H. Schobert B. Richter H.T. Cartailler J.P. Lanyi J.K. J. Mol. Biol. 1999; 291: 899-911Crossref PubMed Scopus (1300) Google Scholar) and bovine rhodopsin (23Palczewski K. Kumasaka T. Hori T. Behnke C.A. Motoshima H. Fox B.A. Le T.I. Teller D.C. Okada T. Stenkamp R.E. Yamamoto M. Miyano M. Science. 2000; 289: 739-745Crossref PubMed Scopus (5003) Google Scholar, 24Li J. Edwards P.C. Burghammer M. Villa C. Schertler G.F. J. Mol. Biol. 2004; 343: 1409-1438Crossref PubMed Scopus (672) Google Scholar) show a few structurally well defined lipids that interact with rhodopsin helices. Molecular dynamic simulations reported existence of specific sites on the surface of rhodopsin for specific interaction with polyunsaturated chains, saturated chains, and cholesterol (25Grossfield A. Feller S.E. Pitman M.C. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 4888-4893Crossref PubMed Scopus (227) Google Scholar). While those observations are very important, they should be verified by results from a method that explores such interactions in fluid bilayers at physiologically relevant conditions. Recently, we demonstrated the feasibility of investigating lipid-rhodopsin interaction by saturation transfer NMR (ST-NMR) in combination with magic angle spinning (MAS) (26Soubias O. Gawrisch K. J. Am. Chem. Soc. 2005; 127: 13110-13111Crossref PubMed Scopus (57) Google Scholar). A preliminary account of saturation transfer by selective excitation between the protons of rhodopsin and lipids, without application of MAS, was presented earlier by Deese et al. (27Deese A.J. Dratz E.A. Hymel L. Fleischer S. Biophys. J. 1982; 37: 207-216Abstract Full Text PDF PubMed Scopus (38) Google Scholar). In ST-NMR, membrane protein resonances are selectively saturated via radiofrequency (rf) pulses. Magnetization is then transferred from the protein to a first layer of lipids surrounding the protein via 1H-1H dipolar contacts allowing identification of the lipid species that are interacting with the protein. In this study, lipid-rhodopsin interactions were investigated by the results on disks and on model of rhodopsin in the spinning the photointermediates responsible for lipid were and as well as selective transfer of magnetization was via a transfer of magnetization to lipid In rhodopsin was into of monounsaturated lipids. The experiments that lipids with polyunsaturated docosahexaenoyl and monounsaturated chains interact specifically with different sites on the surface of rhodopsin. membranes were from rod outer (ROS) of bovine as B.J. 1982; PubMed Scopus Google Scholar). were at and were in the or dim the NMR the disks were into the 50 of or by of by The phospholipids and were by Lipids lipids were at in as in or with the at a of was in a to Rhodopsin was from bovine that were by Litman B.J. 1982; PubMed Scopus Google Scholar). Rhodopsin in % a of or in the were used for rhodopsin with lipids, the phospholipids in were in a of in a The rhodopsin was to the lipid to with an of and a of The was then by into PIPES, 50 with in the formation of was to that the concentration was not than is well the of The was then a of with a of in of bilayers the Gawrisch K. 2004; PubMed Scopus Google Scholar). All but a bilayer the as well as in were by of with of at a of Gawrisch K. 2004; PubMed Scopus Google Scholar). The was and the into with a The were transferred into an for MAS of on NMR experiments on disks were conducted on a NMR with a The 1H were in a MAS at a MAS spinning of with a 1H length of with a of were A of 50 length at an of for selective saturation of protein resonances without lipids. A saturation length of was used if not The of to a chemical of in the in the spinning MAS were for with light from a light that the saturation was and the length of the with reported experiments (26Soubias O. Gawrisch K. J. Am. Chem. Soc. 2005; 127: 13110-13111Crossref PubMed Scopus (57) Google Scholar). were to a that magic angle spinning in of the saturation such that different from of the spinning Current saturation of lipid resonances that is not to the lipid matrix from rhodopsin. The NMR were with a at MAS with a length of and a sequence time of A of at a 1H of was a that was on the from to for the from spinning from application of the of the was with a The the spinning was by of the chemical of the as well as by of lipids. on experiments were on a NMR with a and a MAS probe at a MAS of and a 1H length of The MAS a of for were to the experiments conducted at T. were for was via a of into the of the MAS and had The was as 1H NMR on interaction with saturated and hydrocarbon chains of phospholipids was investigated in bovine to the disks are of phospholipids containing polyunsaturated chains of and saturated chains and Z. Biophys. 1998; PubMed Scopus Google Scholar). Rhodopsin of membrane proteins in disks Biochemistry. PubMed Scopus Google Scholar). A and show the 1H NMR of disks at a MAS spinning of without and with selective saturation of The at from the protons of between the double bonds in polyunsaturated chains the at is by protons from and acid with a from fatty other than DHA and a from of rhodopsin. The of the to resonances very well with the reported fatty acid content of The saturation saturation and saturation for were in experiments on disks and on recombinant with and without rhodopsin. of lipid resonances in membrane was observed at with a of or at a to a chemical of or for up to an of lipid resonances was observed for membranes containing at at a of increased with up to A saturation time of a of at a to a chemical of for selective saturation of rhodopsin without lipid The saturation of rhodopsin in in an of the by a of the at saturated was by a of is defined as of the without and with established that resonances had the time of that in are not responsible for the of the DHA NMR on bovine membranes are of mol % PC, mol % PE, mol % and mol % PS R.E. PubMed Scopus Google Scholar). The MAS NMR with a and a time between of to of to the of is in angle spinning the from the NMR of chemical to a and that are from the by of the MAS spinning In the MAS is spinning be and the of PC, PE, and PS resonances composition of the were assigned by with of model membranes with composition A. Biochemistry. PubMed Scopus Google Scholar). The to the a of that and PE are likely to the NMR chemical this is in with Rhodopsin interaction with specific lipid species was investigated by NMR with of transfer via dipolar from the protons of lipid and headgroups to the lipid as well as via dipolar between rhodopsin protons and lipid It was reported that the protons of and of the of headgroups are the to dipolar interaction with the K. Gawrisch K. Biophys. 75: Scholar, P. Gawrisch K. H. Biophys. 1984; Scopus Google Scholar). the saturation time of magnetization is by the of lipids K. Gawrisch K. J. Phys. Chem. B. 1999; 103: Scopus Google Scholar). the of the is likely to interactions of lipid with sites on rhodopsin. the without and with of rhodopsin be PC and PE resonances are rhodopsin protons are that phospholipids are interacting with rhodopsin. the of PE is than the of PC are for PE for that magnetization transfer to PE with of the PS was for a of magnetization of effect of on interaction of DHA and saturated chains with rhodopsin was by the 1H of disks as a function of time Rhodopsin was the of by the spinning for with the light of a light the MAS from the of the light was as by the of chemical of the were at and the to the of DHA over of DHA was of DHA increased to a of and over to the of The DHA could be to is the of the time of formation of the with increased and the time of a of and a of increased at to a of the of rhodopsin and at 50 to an similar to disks The was on with similar of rhodopsin in the was by with experiments on that were for in the of a into the MAS NMR those results the of the was to the time increased to and increased to and a of DHA not with of the from saturated chains at increased by is not A on membranes without conducted at not time of DHA that the increased of the DHA results from of rhodopsin and not from of or of a a few milliseconds a and equilibrium between and Meta-II is the of DHA of rhodopsin had a of that and Meta-II rhodopsin similar interaction with DHA chains. The changes from Meta-III or The was by DHA over The of rhodopsin with highest magnetization transfer DHA with a time of as it had reported for the E. K. M. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). The of DHA was from DHA by rhodopsin. The that Meta-III is by a magnetization transfer to DHA was by the at a and a the the Meta-III at a was by the of of DHA of Meta-III at the is as first that to the of the at of the DHA of rhodopsin in the is very as well not of at different do not Meta-III at a of to a of Meta-III should be formed the equilibrium is Meta-II. The of Meta-III was by the of a in DHA on of polyunsaturated (DHA) and monounsaturated lipid-rhodopsin experiments on membranes of of with or or were of the saturated and acid chains a the at is from the monounsaturated oleic acid the at is from DHA magnetization transfer between lipids. of the of poly- to monounsaturated lipids in the lipid of hydrocarbon chains were at a of and hydrocarbon chains, of interactions a of chains, the rates of magnetization transfer the lipid matrix by a of without rates of transfer of magnetization by in the of at Gawrisch K. J. Am. Chem. Soc. 2000; Scopus Google Scholar) are of than rates of lipid Gawrisch K. Biophys. J. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). is then to of lipid-rhodopsin interaction without of results from magnetization transfer between lipids. a the rates of magnetization transfer from rhodopsin to DHA or chains are well In experiments conducted as a function of the mol of polyunsaturated lipids, the of the DHA increased between mol % of polyunsaturated lipids in the and saturation a concentration of 50 mol % The of DHA on the mol of polyunsaturated species was the for and mixtures. the of DHA from for to for and to for In contrast, at up to a concentration of polyunsaturated lipid in the and at DHA The in at specific interaction of poly- and monounsaturated lipids with sites on rhodopsin. The concentration has the of with a of of at of polyunsaturated lipids. of the rhodopsin for interaction with polyunsaturated DHA is such that of sites are at 50 mol % polyunsaturated lipid in the sites for polyunsaturated a as a in is Furthermore, if poly- and monounsaturated lipids magnetization from the on then the of DHA should by a of as by a in and at of polyunsaturated lipid that polyunsaturated lipids (DHA) and monounsaturated lipids magnetization from different sites on rhodopsin. DHA was to a model of DHA and lipid It was that rhodopsin has a of sites for of and monounsaturated lipids. of sites is by the of to and and are the mol of DHA and lipids in the The and the of sites for DHA and lipids on and and are the The were for and and the and by the to the It was that to the of and The of the was by an function defined as the of of between and the at were a to account for the of at of DHA and lipids, A for was that the of for of are summarized in for the that in of DHA or as a function of concentration could from changes in that with a of lipid composition, we on and the influence of on The lipid magnetization as a function of time was by the in is the DHA or the magnetization at time and the of saturated magnetization by the at the of is then The of as a function of time could be well with the in the of and of on the of was to changes of not the of of with a to a greater those do not as fast The is that at rates of changes very at changes of the of lipids with docosahexaenoic acid hydrocarbon chains and PE headgroups for rhodopsin In this the lipid-rhodopsin interaction with for lipid species was investigated by rates of magnetization transfer were observed to PE lipids with DHA hydrocarbon chains. All rhodopsin photointermediates transferred magnetization preferentially to DHA-containing lipids, but Meta-III rhodopsin the The of lipid was studied on rhodopsin in lipid of and lipids. It was that polyunsaturated DHA and monounsaturated hydrocarbon chains associate specifically with a sites on rhodopsin. The PE increased rates of magnetization transfer to DHA chains but not of lipid The a rhodopsin model with a lipid-rhodopsin and lipids that interact specifically with sites on the protein surface. NMR the 1H-1H dipolar interactions the magnetization exchange at the lipid-protein In other the rates of magnetization transfer are on the between rhodopsin and lipid the orientation to the of the those and on correlation of lipids in the chains interact with the protein or interact with a of sites and the resonances be A few of the be as not the of lipid composition in the first layer surrounding the protein. are protein sites from magnetization transfer to lipids is it is likely that lipids interact with a of protein sites are at an Furthermore, the not the of lipid-rhodopsin of lipid from the protein surface are of as as they are such that lipids the protein surface the time of experiments conducted as a function of rates of magnetization transfer with that rates of magnetization transfer by 1H-1H dipolar interactions at the lipid-protein are the and not the with A of lipid-rhodopsin interaction experiments as a function of lipid composition as reported in this the not identification of sites on the protein lipids The be by of the protein. with the 1H on a of the DHA with the of saturated chains was or interactions between DHA hydrocarbon chains and rhodopsin with interaction with and acid chains. of that DHA and magnetization from on rhodopsin reported as poly- and monounsaturated sites is by a for a particular lipid. at of and monounsaturated of the polyunsaturated sites are by DHA The monounsaturated sites had this not into rates of magnetization transfer to chains. The saturation of DHA chains, in particular to a with a PE likely that DHA into the rhodopsin as suggested by simulations S.E. Gawrisch K. J. Am. Chem. Soc. 2003; PubMed Scopus Google Scholar). It was observed that hydrocarbon chains with double bonds by do well to the of S.E. Gawrisch K. J. Am. Chem. Soc. 2003; PubMed Scopus Google Scholar). rates of magnetization transfer to PE were in the experiments with and in the 1H experiments on DHA in lipid mixtures. The experiments that interact with rhodopsin that are in the first lipid layer surrounding the protein. experiments not such a preference for interaction with a particular lipid species Biophys. 1998; PubMed Scopus Google Scholar). the investigated by lipid not by composition of hydrocarbon chains. the of the PE from a interaction of DHA chains with rhodopsin in combination with the content of polyunsaturated chains in PE. It was reported that of fatty in PE are DHA in PC Dratz E.A. Biophys. PubMed Scopus Google Scholar). the of lipid headgroups rates of magnetization not the properties of DHA interaction as an and a of it be that the headgroups by allowing a of DHA to sites on rhodopsin or by the of interaction are by the DHA interaction sites were reported by et al. (25Grossfield A. Feller S.E. Pitman M.C. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 4888-4893Crossref PubMed Scopus (227) Google Scholar) on a DHA with of magnetization transfer from rhodopsin photointermediates to DHA and saturated chains was investigated on bovine disks the spinning MAS The has that rates of magnetization transfer to DHA chains are of the of of the DHA a on the time was of DHA a with reported rates of Meta-III was Furthermore, the effect was observed at that formation. the results by et al. R. M. Biochemistry. 2004; PubMed Scopus Google Scholar) and et al. K. E. M. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar) that Meta-III is formed from The rates of magnetization transfer to Meta-III that rhodopsin in Meta-III are for The of saturated hydrocarbon chains had such of on that interaction with the polyunsaturated DHA is that magnetization transfer to polyunsaturated DHA chains is than to monounsaturated or saturated chains. Furthermore, lipid headgroups rates of magnetization transfer from rhodopsin to DHA with highest rates observed for PE. existence of sites for interactions with polyunsaturated and monounsaturated chains at the lipid-rhodopsin The of chain the the the