Effect of packing density on rhodopsin stability and function in polyunsaturated membranes

Rod outer segment disk membranes are densely packed with rhodopsin. The recent notion of raft or microdomain structures in disk membranes suggests that the local density of rhodopsin in disk membranes could be much higher than the average density corresponding to the lipid/protein ratio. Little is known about the effect of high packing density of rhodopsin on the structure and function of rhodopsin and lipid membranes. Here we examined the role of rhodopsin packing density on membrane dynamic properties, membrane acyl chain packing, and the structural stability and function of rhodopsin using a combination of biophysical and biochemical techniques. We reconstituted rhodopsin into large unilamellar vesicles consisting of polyunsaturated 18:0,22:6n3PC, which approximates the polyunsaturated nature of phospholipids in disk membranes, with rhodopsin/lipid ratios ranging from 1:422 to 1:40. Our results showed that increased rhodopsin packing density led to reduced membrane dynamics revealed by the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene, increased phospholipid acyl chain packing, and reduced rhodopsin activation, yet it had minimal impact on the structural stability of rhodopsin. These observations imply that densely packed rhodopsin may impede the diffusion and conformational changes of rhodopsin, which could reduce the speed of visual transduction.     

13C NMR studies of the thermal properties of a model high density lipoprotein. Apolipoprotein A-I-dimyristoylphosphatidylcholine complex

The most abundant lipid and protein components of human plasma high density lipoproteins are phosphatidylcholine and apolipoprotein A-I (A-I). Under appropriate conditions, A-I spontaneously associates with dimyristoylphosphatidylcholine (DMPC) to quantitatively form a lipid-protein complex with a DMPC/A-I molar ratio of 100:1. Differential scanning calorimetry of this complex reveals two broad thermal transitions centered at approximately 27 and 72 degrees C. 13C NMR spectra of the complex have been obtained above, at, and below the lower transition temperature. The 13C resonance arising from the 3' carbon of the fatty acyl chains is a doublet, split by approximately 0.2 ppm, suggesting that the 3' carbon nuclei occupy two magnetically inequivalent sites. By replacing the sn-2 fatty acyl chain with myristate selectively 13C-enriched at carbon 3', we have shown that the splitting is, in fact, a result of magnetic inequivalence of the two sites and have assigned the lower field resonance to the 3' carbon nucleus of the sn-2 chain. The temperature dependence of the NMR relaxation rates indicates that the endothermic transition at 27 degrees C is associated with increased motional freedom for the phospholipids within this complex. The temperature dependence of the fatty acyl chain methylene 13C chemical shifts suggests that the population of gauche conformers increases above the transition temperature. These dynamic and conformational changes are characteristic of gel----liquid crystalline phase transitions observed in pure phospholipid systems. For the DMPC-A-I complex at 37 degrees C, the chemical shifts of the fatty acyl C 4'- 11' methylene envelope and of the C 7' and C 13' resonances occur significantly downfield from the corresponding chemical shifts for the DMPC vesicle. These results suggest that the apoprotein rigidifies the acyl chains by increasing their number of trans conformers.     

Affinity of phosphatidylcholine molecular species for the bovine phosphatidylcholine and phosphatidylinositol transfer proteins. Properties of the sn-1 and sn-2 acyl binding sites

Both the phosphatidylcholine transfer protein (PC-TP) and the phosphatidylinositol transfer protein (PI-TP) act as carriers of phosphatidylcholine (PC) molecules between membranes. To study the structure of the acyl binding sites of these proteins, the affinity of 32 distinct natural and related PC molecular species was determined by using a previously developed fluorometric competition assay. Marked differences in affinity between species were observed with both proteins. Affinity vs lipid hydrophobicity (determined by reverse-phase HPLC) plots displayed a well-defined maximum indicating that the acyl chain hydrophobicity is an important determinant of binding of a phospholipid molecule by these transfer proteins. However, besides the overall lipid hydrophobicity, steric properties of the individual acyl chains contribute considerably to the affinity, and PC-TP and PI-TP respond differently to modifications of the acyl chain structure. The affinity of PC-TP increased steadily with increasing unsaturation of the sn-2 acyl moiety, resulting in high affinity for species containing four and six double bonds in the sn-2 chain, whereas the affinity of PI-TP first increased up to two to three double bonds and then declined. These data, as well as the distinct effects of sn-2 chain double bond position and bromination, indicate that the sn-2 acyl chain binding sites of the two proteins are structurally quite different. The sn-1 acyl binding sites are dissimilar as well, since variation of the length of saturated sn-1 chain affected the affinity differently. The data are discussed in terms of the structural organization of the sn-1 and sn-2 acyl binding sites of PC-TP and PI-TP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific surface association of avidin with N-biotinylphosphatidylethanolamine membrane assemblies: effect on lipid phase behavior and acyl-chain dynamics.

The interaction of avidin with aqueous dispersions of N-biotinylphosphatidylethanolamines, of acyl chain lengths C(14:0), C(16:0), and C(18:0), was studied by using spin-label electron spin resonance (ESR) spectroscopy, (31)P nuclear magnetic resonance ((31)P NMR) spectroscopy, differential scanning calorimetry, and chemical binding assays. In neutral buffer containing 1 M NaCl, binding of avidin is due to specific interaction with the biotinyl lipid headgroup because avidin presaturated with biotin does not bind. Saturation binding of the protein corresponds to a ratio of 50 lipid molecules per tetrameric avidin. Phospholipid probes spin-labeled at various positions between C-4 and C-14 in the sn-2 chain were used to characterize the effects of avidin binding on the lipid chain dynamics. In the fluid phase, protein binding results in a decrease of chain mobility at all positions of labeling while the flexibility gradient characteristic of a liquid-crystalline lipid phase is maintained. There is no evidence from the spin-label ESR spectra for penetration of the protein into the hydrophobic interior of the membrane. At temperatures corresponding to the gel phase, the lipid chain mobility increases on binding protein. The near constancy in mobility found with chain position, however, suggests that in the gel phase the lipid chains remain interdigitated upon binding avidin. Binding of increasing amounts of avidin results in a gradual decrease of the lipid chain-melting transition enthalpy with only small change in the transition temperature. At saturation binding, the calorimetric enthalpy is reduced to zero. (31)P NMR spectroscopy indicates that protein binding increases the surface curvature of dispersions of all three biotin lipids. The C(14:0) biotin lipid yields isotropic (31)P NMR spectra in the presence of avidin at all temperatures between 10 and 70 degrees C, in contrast to dispersions of the lipid alone, which give lamellar spectra at low temperature that become isotropic at the chain-melting temperature. In the presence of avidin, the C(16:0) and C(18:0) biotin lipids yield primarily lamellar (31)P NMR spectra at low temperature with a small isotropic component; the intensity of the isotropic component increases with temperature, and the spectra narrow and become totally isotropic at high temperature, in contrast to dispersions of the lipids alone, which give lamellar spectra in the fluid phase. The binding of avidin therefore reduces the cooperativity of the biotin lipid packing, regulates the mobility of the lipid chains, and enhances the surface curvature of the lipid aggregates. These effects may be important for both lateral and transbilayer communication in the membrane.     

Manipulation of cholesterol levels in rod disk membranes by methyl-beta-cyclodextrin: effects on receptor activation

The effect of cholesterol on rod outer segment disk membrane structure and rhodopsin activation was investigated. Disk membranes with varying cholesterol concentrations were prepared using methyl-beta-cyclodextrin as a cholesterol donor or acceptor. Cholesterol exchange followed a simple equilibrium partitioning model with a partition coefficient of 5.2 +/- 0.8 in favor of the disk membrane. Reduced cholesterol in disk membranes resulted in a higher proportion of photolyzed rhodopsin being converted to the G protein-activating metarhodopsin II (MII) conformation, whereas enrichment of cholesterol reduced the extent of MII formation. Time-resolved fluorescence anisotropy measurements using 1,6-diphenyl-1,3,5-hexatriene showed that increasing cholesterol reduced membrane acyl chain packing free volume as characterized by the parameter f(v). The level of MII formed showed a positive linear correlation with f(v) over the range of 4 to 38 mol % cholesterol. In addition, the thermal stability of rhodopsin increased with mol % of cholesterol in disk membranes. No evidence was observed for the direct interaction of cholesterol with rhodopsin in either its agonist- or antagonist-bound form. These results indicate that cholesterol mediates the function of the G protein-coupled receptor, rhodopsin, by influencing membrane lipid properties, i.e. reducing acyl chain packing free volume, rather than interacting specifically with rhodopsin.     

Protein-lipid interactions at membrane surfaces: a deuterium and phosphorus nuclear magnetic resonance study of the interaction between bovine rhodopsin and the bilayer head groups of dimyristoylphosphatidylcholine

Rhodopsin, isolated from bovine retinal rod outer segment disk membranes, has been reconstituted into bilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine which was deuterated in the terminal methyl groups of the choline polar head group. By use of a mixed detergent system of cholate and octyl glucoside to solubilize the phospholipid and rhodopsin, 15 membrane complexes of predetermined phospholipid to rhodopsin mole ratios of between 350:1 and 65:1 have been produced by exhaustive dialysis and studied by a variety of techniques. Electron micrographs of replicas from freeze-fractured membrane complexes showed that the majority of the lipid, for all rhodopsin:phospholipid ratios, was contained in large bilayer vesicles with diameters in excess of 400 nm. Complexes produced with rhodopsin from frozen retina produced an absorption maximum at 478 nm after photobleaching whereas rhodopsin from fresh retina could be bleached more completely to an absorption maximum at 380 nm. Deuterium nuclear magnetic resonance (NMR) spectra from the lipid head groups of bilayers above the gel to liquid-crystalline phase transition temperature were shown to be sensitive in a systematic way to the presence of rhodopsin which could be bleached to 380 nm. The measured quadrupole splittings, taken as the separation of the turning points of the recorded NMR spectra, decreased from a value of 1.28 kHz for protein-free bilayers to approximately 0.40 kHz for bilayers containing 65 molecules of phospholipid for each rhodopsin at 32 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Raman spectroscopic study of polycrystalline mono- and polyunsaturated 1-eicosanoyl-d(39)-2-eicosenoyl-sn-glycero-3-phosphocholines: bilayer lipid clustering and acyl chain order and disorder characteristics

Polycrystalline lipid samples of a series of mono- and polyunsaturated, double bond positional isomers of 1-eicosanoyl-d(39)-2-eicosenoyl-sn-glycero-3-phosphocholines [C(20-d(39)):C(20:1 Delta(j))PC, with j = 5, 8, 11, or 13; C(20-d(39)):C(20:2 Delta(11,14))PC; and C(20-d(39)):C(20:3 Delta(11, 14,17))PC] were investigated using vibrational Raman spectroscopy to assess the acyl chain packing order-disorder characteristics and putative bilayer cluster formation of the isotopically differentiated acyl chains. Perdeuteration of specifically the saturated sn-1 acyl chains for these bilayer systems enables each chain's intra- and intermolecular conformational and organizational properties to be evaluated separately. Various saturated chain methylene CD(2) and carbon-carbon (C&bond;C) stretching mode peak height intensity ratios and line width parameters for the polycrystalline samples demonstrate a high degree of sn-1 chain order that is unaffected by either the double bond placement or number of unsaturated bonds within the sn-2 chain. In contrast, the unsaturated sn-2 chain spectral signatures reflect increasing acyl chain conformational disorder as either the cis double bond is generally repositioned toward the chain terminus or the number of double bonds increases from one to three. The lipid bilayer chain packing differences observed between the sn-1 and sn-2 chains of this series of monounsaturated and polyunsaturated 20 carbon chain lipids suggest the existence of laterally distributed microdomains predicated on the formation of highly ordered, saturated sn-1 chain clusters.     

Structure and dynamics of plasmalogen model membranes containing cholesterol: a deuterium NMR study

Deuterium nuclear magnetic resonance (2H-NMR) was used to investigate the structure and dynamics of the sn-2 hydrocarbon chain of semi-synthetical choline and ethanolamine plasmalogen in bilayers containing 0, 30, and 50 mol% cholesterol. The deuterium NMR spectra of the choline plasmalogen yielded well-resolved quadrupolar splittings which could be assigned to the corresponding hydrocarbon chain deuterons. The sn-2 acyl chain was found to adopt a similar conformation as observed in the corresponding diacyl phospholipid, however, the flexibility at the level of the C-2 methylene segment of the plasmalogen was increased. Deuterium NMR spectra of bilayers composed of the ethanolamine plasmalogen yielded quadrupolar splittings of the C-2 segment much larger than those of the corresponding diacyl lipids, suggesting that the sn-2 chain is oriented perpendicular to the membrane surface at all segments. Cholesterol increased the ordering of the choline plasmalogen acyl chain to the same extent as in diacyl lipid bilayers. T1 relaxation time measurements demonstrated only minor dynamical differences between choline plasmalogen and diacyl lipids in model membranes.     

Spin-label ESR studies on the interaction of bovine spinal cord myelin basic protein with dimyristoylphosphatidylglycerol dispersions

Electron spin resonance (ESR) spectroscopy and chemical binding assays were used to study the interaction of bovine spinal cord myelin basic protein (MBP) with dimyristoylphosphatidylglycerol (DMPG) membranes. Increasing binding of MBP to DMPG bilayers resulted in an increasing motional restriction of PG spin-labeled at the C-5 atom position in the acyl chain, up to a maximum degree of association of 1 MBP molecule per 36 lipid molecules. ESR spectra of PG spin-labels labeled at other positions in the sn-2 chain showed a similar motional restriction, while still preserving the chain flexibility gradient characteristic of fluid lipid bilayers. In addition, labels at the C-12 and C-14 atom positions gave two-component spectra, suggesting a partial hydrophobic penetration of the MBP into the bilayer. Spectral subtractions were used to quantitate the membrane penetration in terms of the stoichiometry of the lipid-protein complexes. Approximately 50% of the spin-labeled lipid chains were directly affected at saturation protein binding. The salt and pH dependence of the ESR spectra and of the protein binding demonstrated that electrostatic interaction of the basic residues of the MBP with the PG headgroups is necessary for an effective association of the MBP with phospholipid bilayers. Binding of the protein, and concomitant perturbation of the lipid chain mobility, was reduced as the ionic strength increased, until at salt concentrations above 1 M NaCl the protein was no longer bound. The binding and ESR spectral perturbation also decreased as the protein charge was reduced by pH titration to above the pI of the protein at approximately pH 10.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of the lipid acyl chain dynamics between small and large unilamellar vesicles.

13C NMR spin-lattice relaxation (T1) rates and 13C-1H nuclear Overhauser effects (NOEs) were measured in an identical fashion in two lipid preparations having dramatically different curvatures. The T1 times that were obtained at four magnetic field strengths were fit along with the NOEs to simple models for lipid molecular dynamics. The results indicate that phospholipid chain ordering and dynamics are virtually identical in small and large unilamellar vesicles at the time scales sampled by these 13C-NMR studies. The order parameters and reorientational correlation times that characterize the amplitudes and rates of internal acyl chain motions were equal within experimental error for the methylene segments in the middle of the chains. The only significant differences in order parameters and correlation times between the two vesicle types were small and appeared at the ends of the acyl chains. At the carbonyl end the order was slightly higher in small vesicles than large vesicles, and at the methyl end the order was slightly lower for small vesicles. This indicates that in the more planar systems the acyl chains exhibit a slightly flatter order profile than in more highly curved membranes. The use of the same experimental approach in both small and large vesicle systems provided a more reliable and accurate assessment of the effect of curvature on molecular order than has been previously obtained.     

Deciphering the role of individual acyl chains in the interaction network between phosphatidylserines and a single-spanning membrane protein

PMP1 is a small single-spanning membrane protein functioning as a regulatory subunit of the yeast plasma membrane H(+)-ATPase. This protein forms a unique helix and exhibits a positively charged cytoplasmic domain that is able to specifically segregate phosphatidylserines (PSs). A marked groove formed at the helix surface is thought to play a major role in the related lipid-protein interaction network. Mutational analysis and (1)H NMR experiments were therefore performed on a synthetic PMP1 fragment using DPC-d(38) micelles as a membrane-like environment, in the presence of small amounts of POPS. A mutation designed for altering the helix groove was shown to disfavor the POPS binding specificity as much as that affecting the electrostatic interaction network. From POPS titration experiments monitored by a full set of one- and two-dimensional NOESY spectra, the association between the phospholipids and the PMP1 peptide has been followed. Our data reveal that the clustering of POPS molecules is promoted from a stabilized framework obtained by coupling the PMP1 helix groove to a POPS sn-2 chain. To our knowledge, the NOE-based titration plots displayed in this report constitute the first NMR data that directly distinguish the role of the sn-1 and sn-2 acyl chains in a lipid-protein interaction. The results are discussed while taking into account our accurate knowledge of the yeast plasma membrane composition and its ability to form functional lipid rafts.     

Quantifying the differential effects of DHA and DPA on the early events in visual signal transduction

A range of evidence from animal, clinical and epidemiological studies indicates that highly polyunsaturated acyl chains play important roles in development, cognition, vision and other aspects of neurological function. In a number of these studies n3 polyunsaturated fatty acids (PUFAs) appear to be more efficacious than n6 PUFAs. In a previous study of retinal rod outer segments obtained from rats raised on either an n3 adequate or deficient diet, we demonstrated that the replacement of 22:6n3 by 22:5n6 in the n3 deficient rats led to functional deficits in each step in the visual signaling process (Niu et al., 2004). In this study, we examined rhodopsin and phosphodiesterase function and acyl chain packing properties in membranes consisting of phosphatidylcholines with sn-1=18:0, and sn-2=22:6n3, 22:5n6, or 22:5n3 in order to determine if differences in function are due to the loss of one double bond or due to differences in double bond location. At 37 °C the n6 lipid shifted the equilibrium between the active metarhodopsin II (MII) state and inactive metarhodopsin I (MI) state towards MI. In addition, 22:5n6 reduced the rates of MII formation and MII-transducin complex formation by 2- and 6-fold, respectively. At a physiologically relevant level of rhodopsin light stimulation, the activity of phosphodiesterase was reduced by 50% in the 22:5n6 membrane, relative to either of the n3 membranes. Activity levels in the two n3 membranes were essentially identical. Ensemble acyl chain order was assessed with time-resolved fluorescence measurements of the membrane probe diphenylhexatriene (DPH). Analysis in terms of the orientational distribution of DPH showed that acyl chain packing in the two n3 membranes is quite similar, while in the 22:5n6 membrane there was considerably less packing disorder in the bilayer midplane. These results demonstrate that the n3 bond configuration uniquely optimizes the early steps in signaling via a mechanism which may involve acyl chain packing deep in the bilayer.     

Effects of various numbers and positions of cis double bonds in the sn-2 acyl chain of phosphatidylethanolamine on the chain-melting temperature

In an attempt to investigate systematically the effects of various single and multiple cis carbon-carbon double bonds in the sn-2 acyl chains of natural phospholipids on membrane properties, we have de novo synthesized unsaturated C20 fatty acids comprised of single or multiple methylene-interrupted cis double bonds. Subsequently, 15 molecular species of phosphatidylethanolamine (PE) with sn-1 C20-saturated and sn-2 C20-unsaturated acyl chains were semi-synthesized by acylation of C20-lysophosphatidylcholine with unsaturated C20 fatty acids followed by phospholipase D-catalyzed base-exchange reaction in the presence of excess ethanolamine. The gel-to-liquid crystalline phase transitions of these 15 mixed-chain PE, in excess H2O, were investigated by high resolution differential scanning calorimetry. In addition, the energy-minimized structures of these sn-1 C20-saturated/sn-2 C20-unsaturated PE were simulated by molecular mechanics calculations. It is shown that the successive introduction of cis double bonds into the sn-2 acyl chain of C(20):C(20)PE can affect the gel-to-liquid crystalline phase transition temperature, Tm, of the lipid bilayer in some characteristic ways; moreover, the effect depends critically on the position of cis double bonds in the sn-2 acyl chain. Specifically, we have constructed a novel Tm diagram for the 15 species of unsaturated PE, from which the effects of the number and the position of cis double bonds on Tm can be examined simultaneously in a simple, direct, and unifying manner. Interestingly, the characteristic Tm profiles exhibited by different series of mixed-chain PE with increasing degree of unsaturation can be interpreted in terms of structural changes associated with acyl chain unsaturation.     

Characterization of the myristoyl lipid modification of membrane-bound GCAP-2 by 2H solid-state NMR spectroscopy

Guanylate cyclase-activating protein-2 (GCAP-2) is a retinal Ca2+ sensor protein. It is responsible for the regulation of both isoforms of the transmembrane photoreceptor guanylate cyclase, a key enzyme of vertebrate phototransduction. GCAP-2 is N-terminally myristoylated and full activation of its target proteins requires the presence of this lipid modification. The structural role of the myristoyl moiety in the interaction of GCAP-2 with the guanylate cyclases and the lipid membrane is currently not well understood. In the present work, we studied the binding of Ca2+-free myristoylated and non-myristoylated GCAP-2 to phospholipid vesicles consisting of dimyristoylphosphatidylcholine or of a lipid mixture resembling the physiological membrane composition by a biochemical binding assay and 2H solid-state NMR. The NMR results clearly demonstrate the full-length insertion of the aliphatic chain of the myristoyl group into the membrane. Very similar geometrical parameters were determined from the 2H NMR spectra of the myristoyl group of GCAP-2 and the acyl chains of the host membranes, respectively. The myristoyl chain shows a moderate mobility within the lipid environment, comparable to the acyl chains of the host membrane lipids. This is in marked contrast to the behavior of other lipid-modified model proteins. Strikingly, the contribution of the myristoyl group to the free energy of membrane binding of GCAP-2 is only on the order of -0.5 kJ/mol, and the electrostatic contribution is slightly unfavorable, which implies that the main driving forces for membrane localization arises through other, mainly hydrophobic, protein side chain-lipid interactions. These results suggest a role of the myristoyl group in the direct interaction of GCAP-2 with its target proteins, the retinal guanylate cyclases.     

Role of sn-1-saturated,sn-2-polyunsaturated phospholipids in control of membrane receptor conformational equilibrium: effects of cholesterol and acyl chain unsaturation on the metarhodopsin I in equilibrium with metarhodopsin II equilibrium.

The effect of phospholipid bilayer acyl chain packing free volume on the equilibrium concentration of the form of photolyzed rhodopsin which initiates visual signal transduction, metarhodopsin II (meta II), is examined in reconstituted systems formed from the saturated phospholipid dimyristoylphosphatidylcholine (DMPC) and in the polyunsaturated phospholipid sn-1-palmitoyl-sn-2-arachidonoylphosphatidylcholine (PAPC) with and without 30 mol% cholesterol. The extent of meta II formation is determined from both flash photolysis measurements and rapidly acquired absorbance spectra. Equilibrium and dynamic properties of the lipid bilayer are characterized by the dynamic fluorescence properties of 1,6-diphenyl-1,3,5-hexatriene (DPH). DPH orientational properties are characterized by fv, a parameter which reflects the volume available for probe reorientation in the bilayer, relative to that available in an unhindered, isotropic environment [Straume, M., & Litman, B. J. (1987) Biochemistry 26, 5121-5126]. The metarhodopsin I in equilibrium with meta II equilibrium constant, Keq has a linear relationship with fv for rhodopsin in PAPC vesicles with and without cholesterol as well as for rhodopsin in DMPC vesicles, and these two correlation lines have different slopes. The correlations between Keq and fv in PAPC and DMPC systems are compared with a similar correlation in the native rod outer segment disk membrane and one reported previously in an egg phosphatidylcholine (egg PC) system [Mitchell, D. C., Straume, M., Miller, J. L., & Litman, B. J. (1990) Biochemistry 29, 9143-9149].(ABSTRACT TRUNCATED AT 250 WORDS)     

Dynamics of lipid chain attached fluorophore 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) in negatively charged membranes determined by NMR spectroscopy

We have determined the average location and dynamic reorientation of the fluorophore 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) attached to a C12 sn-2 chain of a phosphatidylserine (PS) analogue (C12-NBD-PS) in zwitterionic phosphatidylcholine (PC) and negatively charged phosphatidylserine (PS) host membranes. (1)H magic angle spinning nuclear Overhauser enhancement spectroscopy indicates a highly dynamic reorientation of the aromatic molecule in the membrane. The average location of NBD is characterized by a broad distribution function along the membrane director with a maximum indicating the location of the probe in the lipid/water interface of the lipid membrane. This behavior can be explained by a backfolding of the sn-2 chain towards the aqueous phase. Small differences in the distribution profiles of the NBD group along the membrane normal between PC and PS host membranes were found: in a PC host membrane, the NBD distribution has its maximum in the glycerol region; in a PS host membrane, NBD resides mostly in the upper chain region. These differences may be accounted for by packing differences in the PC versus PS host membranes. As seen by (2)H NMR order parameters, PS bilayers show a much higher packing density compared to PC membranes. Consequently, backfolding of the sn-2 chain with the NBD group attached causes a larger decrease of molecular order of the sn-1 chain in PS than in PC membranes. The broad distributions obtained for lipid chain attached NBD molecules reflect the motional freedom and molecular disorder in the liquid-crystalline lipid membrane.     

Packing characteristics of highly unsaturated bilayer lipids: Raman spectroscopic studies of multilamellar phosphatidylcholine dispersions

The thermotropic properties and acyl chain packing characteristics of multilamellar dispersions of highly unsaturated lipids were examined by Raman spectroscopy. Bilayer assemblies were composed of POPC (1-palmitoyl-2-oleoylphosphatidylcholine), PAPC (1-palmitoyl-2-arachidonylphosphatidylcholine), and PDPC (1-palmitoyl-2-docosahexaenoylphosphatidylcholine), lipid systems possessing saturated sn-1 chains and unsaturated sn-2 chains with one, four, and six double bonds, respectively. Raman spectra were recorded in the acyl chain 2800-3100-cm-1 carbon-hydrogen (C-H) stretching and 1100-1200-cm-1 carbon-carbon (C-C) stretching mode regions, spectral intervals reflecting both the inter- and intrachain order/disorder properties of the various lipid dispersions. In order to obtain C-H stretching mode spectra relevant solely to the sn-1 chains of PAPC and PDPC, liquid-phase spectra of arachidonic and docosahexaenoic acid, respectively, were subtracted from the observed phospholipid spectra. The unsaturated sn-2 chains of PAPC and PDPC undergo minimal conformational reorganizations as the bilayers pass from the gel to liquid-crystalline phases. Phase transition temperatures, Tm, derived from statistically fitting the temperature-dependent Raman spectral data are approximately -2.5, -22.5, and -3 degrees C for POPC, PAPC, and PDPC, respectively. As the degree of unsaturation increases from POPC to PAPC and PDPC, the cooperativity of the phase transition, as measured by its breadth, decreases. Estimates of the transition widths from the temperature profiles are approximately 15 degrees C for PAPC and 20 degrees C for PDPC. The behavior of various Raman spectral parameters for the lipid gel phase reflects the formation of lateral microdomains, or clusters, whose packing properties maximize the van der Waals interactions between sn-1 chains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural aspects of pressure effects on infrared spectra of mixed-chain phosphatidylcholine assemblies in D2O

The barotropic behavior of D2O dispersions of 1-stearoyl-2-caproyl-sn-glycero-3-phosphocholine, C(18):C(10)PC, a highly asymmetric phospholipid in which the length of the fully extended acyl chain at the sn-1 position of the glycerol backbone is twice as long as that at the sn-2 position, has been investigated by high-pressure Fourier transform infrared spectroscopy. This asymmetric phosphatidylcholine bilayer at room temperature displays a pressure-induced phase transition corresponding to the liquid-crystalline----gel phase transition at 1.4 kbar. A conformational ordering of the lipid acyl chains is observed to take place abruptly at the transition pressure of 1.4 kbar. However, the lamellar lipid molecules and their acyl chains remain to be orientationally disordered in the gel phase until the applied pressure reaches 5.5 kbar. In the gel phase of fully hydrated C(18):C(10)PC, the asymmetric lipid molecules assemble into mixed interdigitated bilayers with perpendicular orientation of the zigzag planes among neighboring acyl chains. The role of excess water played in the interchain structure and the behavior of excess water and bound water under high pressure are also discussed.     

Conformation of fatty acyl chains in alpha- and beta-phosphatidylcholine and phosphatidylethanolamine derivatives in sonicated vesicles

Mono- and dimethylated derivatives constitute important intermediates in the conversion of phosphatidylethanolamine (PE) to phosphatidylcholine (PC) in eucaryote membranes. 1H-NMR techniques were utilized to examine the conformation of the region of the fatty acyl chains that is close to the polar group in the series of alpha-phospholipids: PE, N-methyl-PE, N,N-dimethyl-PE, and PC. The same series of polar groups, but on phospholipid containing sn-1 and/or sn-3 fatty acyl chains (beta-phospholipids) were also examined. All of the phospholipids were in the form of small sonicated vesicles which are widely utilized as membrane models. The alpha-methylene group of the sn-1 and sn-2 fatty acyl chains of the alpha-phospholipids give rise to separate signals due to the non-equivalency of these chains with respect to the glycerol phosphate backbone on all alpha-phospholipids tested. Additionally, differences in the environment of the PC molecules as well as N-methyl-PE, and N,N-dimethyl-PE, but not PE itself on the inside and outside of the vesicles are reflected in the chemical shift of the alpha-methylene protons. On the other hand, all of the beta-phospholipids (including beta-PE) were found to reflect the inside/outside packing differences in their alpha-methylene groups. The bilayer packing does not induce any nonequivalence in the chemically equivalent acyl chains. In mixed micelles with detergents, beta-phospholipids showed one alpha-CH2 signal for all phospholipids. These results are consistent with a common conformational arrangement for the fatty acyl chains in all alpha-phospholipids that have been investigated no matter what aggregated form. The conformational arrangement in the beta-phospholipids is different, but again is similar for all of the compounds tested in various aggregated forms.     

Effect of protein hydration on receptor conformation: decreased levels of bound water promote metarhodopsin II formation.

Neutral solutes were used to investigate the effects of osmotic stress both on the ability of rhodopsin to undergo its activating conformation change and on acyl chain packing in the rod outer segment (ROS) disk membrane. The equilibrium concentration of metarhodopsin II (MII), the conformation of photoactivated rhodopsin, which binds and activates transducin, was increased by glycerol, sucrose, and stachyose in a manner which was linear with osmolality. Analysis of this shift in equilibrium in terms of the dependence of ln(Keq) on osmolality revealed that 20 +/- 1 water molecules are released during the MI-to-MII transition at 20 degrees C, and at 35 degrees C 13 +/- 1 waters are released. At 35 degrees C the average time constant for MII formation was increased from 1.20 +/- 0.09 ms to 1.63 +/- 0.09 ms by addition of 1 osmolal sucrose or glycerol. The effect of the neutral solutes on acyl chain packing in the ROS disk membrane was assessed via measurements of the fluorescence lifetime and anisotropy decay of 1,6-diphenyl-1,3,5-hexatriene (DPH). Analysis of the anisotropy decay of DPH in terms of the rotational diffusion model showed that the angular width of the equilibrium orientational distribution of DPH about the membrane normal was progressively narrowed by increased osmolality. The parameter fv, which is proportional to the overlap between the DPH orientational probability distribution and a random orientational distribution, was reduced by the osmolytes in a manner which was linear with osmolality. This study highlights the potentially opposing interplay between the effect of membrane surface hydration on both the lipid bilayer and integral membrane protein structure. Our results further demonstrate that the binding and release of water molecules play an important role in modulating functional conformational changes for integral membrane proteins, as well as for soluble globular proteins.