Sensing isothermal changes in the lateral pressure in model membranes using di-pyrenyl phosphatidylcholine Richard H. Templer,* SaÜron J. Castle A. Rachael Curran Garry Rumbles and David R. Klug T he Department of Chemistry Imperial College L ondon UK SW 7 2AY Received 17th August 1998 In this work we present data from a homologous series of di-pyrenyl phosphatidylcholine (dipyPC) probes which can sense lateral pressure variations in the chain region of the amphiphilic membrane (lateral pressures are tangential to the interface). The dipyPC has pyrene moieties attached to the ends of equal length acyl chains on a phosphatidylcholine molecule. Ultraviolet stimulation produces both monomer and excimer —uorescence from pyrene. At low dilutions of dipyPC in model membranes the excimer signal is entirely intra-molecular and since it depends on the frequency with which the pyrene moieties are brought into close proximity the relative intensity of the excimer to monomer signal g is a measure of the pressure.We synthesised or purchased dipyPC probes with the pyrene moieties attached to acyl chains having 4 6 8 and 10 carbon atoms and then measured g in fully hydrated bilayers composed of dioleoylphosphatidylcholine and dioleoylphosphatidylethanolamine (DOPC and DOPE respectively). Although the resolution of our measurements of lateral pressure as a function of distance into the monolayer was limited we did observe a dip in the excimer signal in the region of the DOPC/DOPE cis double bond. As we isothermally increased the DOPE composition and hence the desire for interfacial curvature we observed as expected that the net excimer signal increased.However this net increase was apparently brought about by a transfer of pressure from the region around the glycerol backbone to the region near the chain ends with the lateral pressure dropping above the cis double bond but increasing at a greater rate beyond the double bond. (1) gc\12i(c1]c2[2c0)2]iG c1 c2 Introduction In an amphiphilic bilayer the back to back monolayers are restrained to lie —at even though each will in general possess an intrinsic desire for interfacial curvature. The curvature elasticity1 of the monolayer may be expressed in terms of the bending energy per unit area gc 0 2 . where c c and are the principal curvatures of the region of interest c0 is called the spontaneous 1 2 curvature of the interface i is the mean curvature modulus and i is the Gaussian curvature G modulus.For the case where the monolayer is held —at the stored curvature elastic energy is simply 2ic This is precisely the case for cell membranes so we would anticipate that the amphiphilic fabric of the cell membrane would be storing a curvature elastic energy in proportion to i and c0 and it 41 Faraday Discuss. 1998 111 41»53 has been suggested that this stored energy may play a role in modulating the behaviour of membrane proteins.2,3 Evidence that this is the case has been building steadily,4h8 but the correlation between protein activity and the physical state of the membrane has for the most part remained qualitative.In part this has been because our knowledge of i and c for most amphiphilic lipid 0 systems remains sketchy. However even if we had a better and more reliable catalogue of values of i and c we would have to accept that these two parameters are a rather crude attempt to 0 characterise the physical state of the amphiphilic membrane. The connection between the stored curvature elastic energy and a more complete description of the physical state of the monolayer is in principle relatively straightforward. The desire for monolayer curvature arises because of the diÜerential distribution of lateral pressure Fig. 1. At the polar/apolar interface there is a strong inward pressure as the system attempts to limit the contact between water and hydrocarbon.This is resisted in the chain region where there is a strong outward pressure due to thermally driven collisions between chains. In the headgroup region there may also be positive contributions to the lateral pressure from steric hydrational and charge eÜects but also negative contributions may occur if there is direct hydrogen bonding between headgroups. Although the integral of the lateral pressures for the monolayer must necessarily come to zero the –rst moment of the lateral pressure will in general be non-zero. It is this torque tension which represents the monolayerœs frustrated desire for interfacial curvature and it is related to i and c0 by (2) 0 Pl n(z)z dz\2ic 0 0 where l is the monolayerœs thickness9 and we use the convention that c is negative when the 0 monolayer wishes to bend towards the water.Clearly n(z) is the most complete description of the average lateral forces exerted by the lipid membrane on proteins inserted into the bilayer. Unfortunately measurements of c i and cannot uniquely de–ne n(z). In this work we have therefore attempted to take the alternative approach of trying to measure n(z) directly. This is as far as we are aware the –rst attempt to do this that has been reported in the literature. We have restricted ourselves to probing the pressure in the chain region. We have done this for a number of reasons. Statistical mechanical calculations of the lateral pressure pro–les in the chain regions are possible because of the relatively simple nature of the interactions between —uid chains.10h12 This is not true for the headgroup and interfacial regions.Fluorescent probe molecules tend to be relatively bulky and this militates against their use either in the headgroup region or the interface where it is already known that very small changes can have profound eÜects on phase behaviour.3,13 The evidence from the eÜect of small amounts of hydrophobic additives in bilayers indicates that the eÜect on the phase behaviour of having the probe in the —uid chain region would be signi–cantly less.3 Finally since the chains form an extended region we are most likely to be able to extract some of the details of the shape of the pressure pro–le here. Fig. 1 Distribution of lateral pressure within a —at monolayer. The isotropic lateral pressure n can be plotted as a function of depth into the monolayer z.Faraday Discuss. 1998 111 41»53 42 Being an extended region of positive lateral pressure we would expect the magnitude of the pressure and hence the magnitude of variations to be less than in the headgroup and interfacial regions. We may estimate what the magnitude of n(z) might be for the relatively well characterised lipid system we have used in this work; dioleoylphosphatidylcholine (DOPC) and dioleoylphosc phatidylethanolamine (DOPE). For DOPC and DOPE at 25 °C we have 0\[1/90 Aé ~1 and [1/56 Aé ~1 and i\9 kT and 12 kT respectively. The determination of the torque tension is independent of the origin of the integration and hence we choose to place this at the headgroup region so that the torque tension arises predominantly from the pressure in the chain region.We will assume that n(z) in the chain region is constant (\n6 c) and hence that n6 c\[4ic0/l2. Since l is approximately 18 Aé in the case of DOPC and DOPE the average pressure in the chain region for —at monolayers of DOPC and DOPE will be 5 and 10 MPa respectively. These estimates are of the same order of magnitude as the theoretical calculations of Szleifer and co-workers on saturated chains.10 The experiments we present here examine the changes in the lateral pressure pro–le in the chain region of DOPC/DOPE membranes as a function of the lipid composition. Our probes will therefore reside in a milieu where the pressures are at least one order of magnitude greater than atmospheric and where we can vary this pressure by a factor of two by changing the relative composition of DOPC and DOPE in excess water.Our report is concerned with the measurement of n(z) in the —uid lamellar (La) phase which means that we will only brie—y report on our measurements at high DOPE contents where the inverse hexagonal phase is found. We chose to use dipyPCs Fig. 2 to make —uorescence measurements of lateral pressure. In particular we were aiming to make use of intra-molecular pyrene excimer formation as a measure of the lateral pressure. Excimers will occur when a pyrene moiety in its excited state comes into close proximity (B3.5 Aé ) and in the correct orientation (face to face) to a ground state pyrene and they form an excited dimer or excimer.14h19 Using a model in which pyrene re-orientation must occur after aggregation Cheng and co-workers have shown that aggregation is the rate limiting step in excimer formation in dipyPCs.20 Since the frequency with which aggregates form will be proportional to the lateral pressure their –ndings imply that the excimer signal in turn will be Fig.2 The 10dipyPC —uorescent probe molecule. 43 Faraday Discuss. 1998 111 41»53 proportional to the lateral pressure. In practice it is more reliable to measure the ratio of the excimer to the monomer —uorescence intensity g. Although it has not been stated there are in fact a number of reports which show evidence that g is a measure of lateral pressure. In the –rst reports of the synthesis and —uorescence of dipyPC [1,2-bis(1-pyrenyldecanoyl)-L-a-phosphatidylcholine or 10dipyPC] Sunamoto and co-workers showed that low concentrations of 10dipyPC in dipalmitoylphosphatidylcholine and egg lecithin bilayers exhibited increases in g with the addition of cholesterol or increases in temperature.21,22 Both are known to increase the monolayer torque tension.Cheng and co-workers used 1,2-bis(1- pyrenylmyristoyl)-L-a-phosphatidylcholine (14dipyPC) to examine lipids in both the lamellar and inverse hexagonal phases.23 Both g and the monomer lifetime were found to be sensitive to the temperature induced lamellar to inverse hexagonal phase transition in DOPE. Butko and Cheng24 used 14dipyPC to examine mixed dilinoleoylphosphatidylethanolamine/palmitoyloleoylphosphatidylcholine mesophases.Using Birksœ description of the kinetics for excimer formation,14 Fig. 3 they showed that g can be related to the rate constants by (3) g\ (kfD/kfM)kDMC (kD]kMD) For dipyPCs at low –xed concentration the ratio kfD/kfM the ratio between the radiative decay parameters of the excimer and monomer and (kD]kMD)~1 the —uorescence lifetime of the excimer were found to be independent of temperature. Hence g was simply proportional to kDM the rate constant of excimer formation. They used this to determine the activation energy for excimer formation which they found to be lower in the L phase. Again this is consistent with the a increased lateral pressures in the L phase. Sassaroli and co-workers25 found that g for 10dipyPC a decreases with applied hydrostatic pressure in DOPC.They calculated that the activation energy for excimer formation decreased with increasing pressure in agreement with Butko and Chengœs –ndings. Cheng and co-workers have also used dipyrenylphosphatidylethanolamine dipyPE probe molecules.20 In these studies they found that g for 4dipyPE was always greater than g for 10dipyPE at any given DOPC/DOPE composition and temperature. This is consistent with the theoretical calculations of the lateral pressure pro–le where the lateral pressure is always highest nearest the polar/apolar interface.10 Although the data on dipyPCs in bilayers have been the subject of intense analysis there has been as far as we are aware only one mention in the literature of lateral pressures being the source of variation in the photophysical properties of dipyPC.The authors26 have in fact attempted to determine the variation in c for DOPC/DOPE mixtures in a complex analysis of their 0 spectroscopic data. In doing so they have unwittingly made use of the fact that i for DOPC and DOPE are so close that almost all of the variation in torque tension is due to the changes in c0 .27 fD k kfM k and k are the iM kDM is the second-order rate constant for excimer iD Fig. 3 Birksœ kinetic scheme for excimer formation. M is the monomer M* the excited monomer and MM* the excited dimer. and are the monomer and excimer radiative decay parameters monomer and excimer non-radiative decay parameters from the dissociation of the excimer. formation and C is the pyrene concentration.kMD is the rate constant for the regeneration of excited monomer Faraday Discuss. 1998 111 41»53 44 With these encouraging data we have set about to record how the lateral pressure pro–le in the chain region changes as a function of composition in DOPC/DOPE mixtures. and aluminium chloride (approximately 0.2 and 0.4 mol respectively) Materials and methods Materials and synthesis DOPC and DOPE were purchased from Avanti Polar Lipids (Alabaster Alabama USA). They had a stated purity [99% which we con–rmed by thin layer chromatography (TLC) before and after use. We found no degradation of these components in our experiments. Vauhkonen and Somerharju28 have shown that the maximum excimer —uorescence intensity is achieved where the pyrenyl moieties are appended to acyl chains of equal length.DipyPCs having pyrenyl moieties attached to acyl chains with 4 and 10 carbon atoms (4dipyPC and 10dipyPC) were purchased from Molecular Probes Inc. (Eugene Oregon USA). They had a stated purity [99% which we con–rmed with high pressure liquid chromatography (HPLC). Since dipyPCs having chain lengths of 6 and 8 (6 and 8dipyPC) were not available commercially we synthesised these ourselves. Their production was broken into two major parts the synthesis of the pyrenyl fatty acids 6-(pyren-1-yl)hexanoic acid and 8-(pyren-1-yl)octanoic acid ; and their subsequent acylation onto glycerophosphocholine. The –rst part of the synthesis was carried out in our laboratory using a modi–cation of the method described by Sunamoto and co-workers,21 whilst the –nal stage of the synthesis was carried out for us by Dr D.M. Phillips of Lipid Products Ltd. (Nut–eld Surrey UK) using an adaptation of the method used to synthesise 4dipyPC.29 2)nCO2H Making the pyrenyl fatty acid proceeds via a three step synthesis. 0.25 mol EtO C(CH 2 (n\6 or 8) and 0.5 mol thionyl chloride were mixed in 400 ml of low boiling point petroleum ether and left at room temperature until no solid monoethyl adipate remained about 16 h. The petroleum ether and much of the thionyl chloride was removed on a rotary evaporator using toluene to form an azeotropic mixture with the thionyl chloride. The monoethyl adipoyl chloride or monoethyl suberoyl chloride were then distilled under vacuum at 66»70 °C 2 mmHg. The yield varied from 70»90%.The EtO C(CH 2 2)nCOCl were mixed with 65 ml of dichloromethane and placed in an ice bath. Approximately 0.2 mol of pyrene in 225 ml of dichloromethane was added dropwise to the mixture and stirred for 16 h. Iced water (190 ml) was added slowly with stirring to the orange reaction mixture causing a vigorous exothermic reaction. After 16 h of stirring the dark green dichloromethane rich layer was removed and dried with sodium sulfate before removal of the dichloromethane on a rotary evaporator. The remaining product was recrystallised from methanol at a yield of 60»80% with respect to pyrene. Approximately 0.05 mol of 1-(5-carbethoxy-1-oxapentanyl)pyrene or 1-(7-carbethoxy-1-oxaheptanyl) pyrene 0.18 mol of potassium hydroxide and 0.17 mol of hydrazine hydrate were mixed with 210 ml of diethylene glycol and re—uxed at 162»178 °C for 4 h.After re—uxing the excess hydrazine hydrate was removed by distillation at about 100 °C. The remaining mixture was cooled to room temperature and 250 ml water was added dropwise with stirring. The mixture was allowed to stir for 12 h. We found difficulties in obtaining a pure precipitate of the pyrenyl fatty acids. In the case of the 6-(pyren-1-yl)hexanoic acid we ground the product mixture and stirred with several washings of acetone to dissolve it. The addition of hexane was then used to make the acid precipitate. Mass spectroscopy infra-red spectroscopy elemental analysis and TLC indicated that although not pure the sample was predominantly 6-(pyren-1-yl)hexanoic acid.In the case of the 8-(pyren-1-yl)- octanoic acid the potassium salt of the acid had already precipitated out of the reaction mixture. The salt was –nely ground and vigorously stirred with a solution of hydrochloric acid and water for two days resulting in a beige solid which was mostly 8-(pyren-1-yl)octanoic acid. In this case the precipitate was additionally analysed by NMR and though not pure was apparently predominantly composed of 8-(pyren-1-yl)octanoic acid. It was assumed that the 6-(pyren-1-yl)hexanoic acid was 66% pure and was treated with 1,1@-carbonyldiimidazole in dry chloroform for 26 h at 30 °C. After concentrating the solution it 45 Faraday Discuss. 1998 111 41»53 was used to acylate glycerophosphocholine vacuum dried onto a viscose support material at an assumed molar ratio of 6-(pyren-1-yl)hexanoylimidazole glycerophosphocholine of 4 1.The acylation was carried out with stirring at 40 °C for 93 h. The dark orange solution was then chromatographed on Davisil (a silicilic acid) eluting with binary and ternary chloroform methanol water solvent systems. Nitrogen bubblers were used in the receivers. The products were monitored and detected by TLC in chloroform methanol water 69 27 4. The disubstituted phosphatidylcholine ran to R 0.35 the monosubstituted phosphatidylcholine ran to R 0.16. The 6dipyPC F F was not completely pure by TLC with –ve spots seen on the plate which could not be separated by the column. A small amount of the mono-acylated product was also produced.With the assumption that the 8-(pyren-1-yl)octanoic acid was 80% pure the same synthesis was F used to produce 8dipyPC. The reaction mixture was chromatographed as before showing a major spot at R 0.31 due to 8dipyPC and a small contaminant running just ahead. A small amount of F the monosubstituted product was also formed with a R of about 0.11. To purify these products we used preparative HPLC. A solvent system of chloroform methanol water (60 40 7) was used with a silica column (250 mm]9.6 mm). The absorption detector was set to 345 nm and the —ow rate used was 1 cm3 min~1. Approximately 0.2 mg of crude product was put on to the column. 8dipyPC showed a single peak after treatment (purer than the 4 and 10dipyPC) but 6dipyPC showed a secondary contaminant peak.Fractions of each of the peaks were taken and examined under UV light for —uorescence. Only the major fraction exhibited —uorescence and fast atom bombardment mass spectroscopy con–rmed that this was 6dipyPC. In subsequent experiments we determined that the contamination was caused by exposure to the UV light during HPLC. We were able to obtain the same eÜects in all of the dipyPCs but the 6dipyPC appeared to be peculiarly sensitive to such degradation. Other work with pyrene probes suggests that the use of HPLC grade chloroform stabilised with amylenes causes degradation in the presence of light but that reagent grade chloroform does not have the same eÜect.30 The 6dipyPC used for all subsequent work was that obtained after puri–cation by HPLC which we estimate to be better than 90% purity.More importantly perhaps the —uorescence arises entirely from 6dipyPC. All samples were dissolved in reagent grade chloroform stored at [23 °C in the dark and handled in darkroom lighting conditions to avoid light induced sample degradation. Sample preparation and instrumentation Samples were prepared by freeze drying the individual components i.e. DOPC DOPE and dipyPC. With the assumption that each component was in fact dihydrate the requisite masses of DOPC and DOPE were then weighed under a dry nitrogen atmosphere and co-dissolved in cyclohexane. The dipyPC was also dissolved in cyclohexane at a known concentration and a sufficient quantity added such that the –nal concentration of dipyPC to total lipid was 0.1 mol%.In agreement with previous work,20v26,28 we found that at this concentration virtually no intermolecular excimers form. The resulting mixture was then freeze dried and mixed with an excess of fresh triply distilled deionised water. We did not use de-oxygenated water since separate experiments indicated that this did not have a detectable eÜect on the pyrene —uorescence. The mixture was thoroughly homogenised by centrifuging it up and down in a sealed tube. A small amount of sample was then scooped out and placed between two 1 mm thick quartz glass slides separated by a 100 lm thick silicone rubber gasket and sheared until the sample appeared clear and exhibited little if any birefringent textures. The slide was then sealed up along its edges with a high temperature adhesive tape and the —uorescence measurements taken.The silicone rubber gave no —uorescence signal and was not hygroscopic. These samples were robust giving identical results after 24 h and being heatable to 80 °C. In our initial experiments we did not shear the sample to surface align the liquid crystalline phase. We found that as a consequence the intensity of the excimer and monomer —uorescence varied widely with time. We assume that this occurred because defect structures created during homogenisation were being slowly annealed and that the lateral pressures in such highly curved regions were quite diÜerent from —at ones. By aligning the mesophase the number of defects was lowered to levels Faraday Discuss. 1998 111 41»53 46 where we were unable to detect any variation in the intensity of the excimer and monomer —uorescence over a period of 1 h.Most previous studies of dipyPC —uorescence in amphiphilic bilayers have been done with vesicles. These are convenient since they can be made into clear solutions but variations in the vesicle diameter osmotically induced tension31 and fusion events can all aÜect the lateral pressure. For the —uorescence measurements reported here we used a SPEX Fluoromax photon counting —uorimeter running DM3000FL software (Spex Industries Inc. Edison New Jersey USA). Excitation was at 345 nm and the emission spectrum was recorded between 370 and 600 nm in 0.5 nm increments integrating at each wavelength for 0.1 s (the excitation and emission bandwidth were both 0.4 nm).All spectra were corrected for variations in detector response with wavelength. The —at samples were held in a temperature controlled cell at an angle of 22° to the incoming excitation beam. This ensured that virtually no scattered excitation light entered the emission monochromator. Sample temperature during these measurements was controlled to within ^0.03 °C by a thermoelectric heater of our own design. A drift in temperature of 0.1 °C resulted in a 2% change in excimer and monomer —uorescence intensities which were easily detected. In Fig. 4 we show a typical example of the emission spectra from dipyPC in DOPC/DOPE bilayers. There are two sharp and intense monomeric signals at 377 and 397 nm whilst the broad excimer emission peaks at 477 nm.By converting the spectra to energy scales it is possible to –t the excimer peak to a Gaussian curve and thence determine the ratio of the excimer to monomer signal by integrating the remaining monomer signal and the extracted excimer signal. However we found no advantage in doing this over measuring the intensity of the 377 nm peak and the excimer peak intensity. We did not use the 397 nm peak because our signal deconvolution indicated that there was signi–cant overlap between it and the excimer emission. No matter which technique we used we found that sample to sample reproducibility in the measurement of g was ^5%. Results In Fig. 5 we present our measurements of g at 25 °C in DOPC/DOPE membranes in the presence of excess water. Up to 84 mol% DOPE in the binary lipid mixture is in the L phase.Over a a period of several days at room temperature the system will convert to an inverse bicontinuous Fig. 4 Fluorescence emission spectrum from 4dipyPC in the L phase excited at 345 nm. The sharp peaks in a the UV end of the spectrum are due to monomeric pyrene emission whereas the broad peak in the visible region is from pyrene excimers. 47 Faraday Discuss. 1998 111 41»53 Fig. 5 Variation in g at 25 °C as a function of DOPC/DOPE composition in the L phase in excess water. a The excimer to monomer ratio has been measured using 4- 6- 8- and 10dipyPC (Ö ),K L and respectively). Linear –ts to the data are shown and the coefficients are listed in Table 1. cubic phase in the region of the phase boundary.32 Independent checks with polarising microscopy and small angle X-ray diÜraction indicated that this had not occurred for our samples in the region of the phase boundary.We observe both increasing and decreasing signals in the lamellar phase as we increase the amount of DOPE. We have –tted the variation in g to linear functions which are shown in Fig. 5 and reported in Table 1. Cheng and co-workers have also measured g in 4- and 10dipyPC in DOPC/DOPE membranes at 20 and 30 °C.20 In their measurements they used the intensities at 392 and 475 nm to determine g. These are peculiar values since 392 nm does not correspond to a maxima but sits on the rising edge of the second most intense monomer peak. Analysing our results at the same wavelengths we obtain good agreement as far as the rate of change of g with respect to composition is concerned but the absolute values of g disagree by approximately 6%.In fact there is a considerable degree of variation between diÜerent groups in the absolute value of g measured. We have found that the ratio is aÜected by the sample preparation used. For example we were able to achieve as much as a factor of 2 diÜerence in the value of g depending on whether Table 1 Fitting coefficients for the variation of g as a function of DOPC/DOPE composition at 25 °C in the L phase in excess water (see Fig. 5)a a Intercept dipyPC chainlength Gradient/10~3 4 6 8 10 [1.2^0.2 [0.55^0.15 0.5^0.2 1.3^0.1 1.143^0.009 0.788^0.008 0.59^0.01 0.693^0.008 a The data were –tted using the Marquardt» Levenberg algorithm implemented on Kaleidagraph.Faraday Discuss. 1998 111 41»53 48 we aligned our samples between the glass slides or not. Consistent sample preparation methods are therefore critical to obtaining a self consistent set of data. Returning to Fig. 5 one might at –rst glance expect the lateral pressures and hence g to rise at all depths as we increase the monolayer torque tension by adding DOPE. In fact if we sum g at all dipyPC chain lengths we do –nd this Fig. 6. What our measurements indicate is that whilst g is dropping for the 4- and 6dipyPC probes it is rising more steeply for 8- and 10dipyPC. Since we know that DOPE increases the torque tension in DOPC/DOPE bilayers by reducing the average molecular cross-sectional area (72 Aé 2 for DOPC (see Fig.7) and 64.5 Aé 2 at 75 mol% DOPE33) we should similarly see a drop in g for 4dipyPC if we dehydrate DOPC bilayers since this also reduces the average molecular cross-section and hence the net lateral pressure. The results of such an experiment are shown in Fig. 7 where we observe a halving in g for a reduction in molecular area from 74 to 68 because these measurements were made directly on the X-ray capillary samples used to determine molecular dimensions which are not surface aligned.) Aé 2. (The diÜerences in the absolute values of g compared to Fig. 5 have arisen These measurements therefore indicate that in DOPC/DOPE bilayers isothermal increases in torque tension involve not only a net increase in the lateral pressure but also a transfer of pressure towards the chain ends.In Fig. 8 we have sequentially plotted the pro–les of g calculated from our –t to the data in Fig. 5 at 0 40 and 80 mol% DOPE. The transfer of pressure to chain ends is clear and our results indicate that this occurs across a minimum in g. If one assumes that the dipyPC and the DOPC/ DOPE chains contract by the same proportion upon chain melting then this minimum is in the neighbourhood of the carbon double bond (the pyrene moieties span the C9»C15 positions of DOPC and DOPE when they are in the all-trans conformation). Such a dip in the lateral pressure has not been predicted in the case of saturated chains but recent calculations for unsaturated chains show a dip in the order parameter at the position of the double bond.34 This lends credence to the idea that the dip in the pro–le of g is due to a dip in the lateral pressure.We have measured g in the inverse hexagonal phase as a means of providing us with further evidence that we are recording changes in the lateral pressure pro–le. We would expect that the lateral pressure and hence g would drop at all points between the pivotal surface and the chain ends. Using Chen and Randœs data on the pivotal surface in DOPE27 and Reiss-Husson and Luzzatiœs data for the Fig. 6 Variation in the total excimer to monomer signal gnet at 25 °C as a function of DOPC/DOPE composition in excess water. The total excimer to monomer signal is found by summing the individual values of g from 4- 6- 8- and 10dipyPC. 49 Faraday Discuss.1998 111 41»53 Fig. 7 Changes in unit cell dimensions (L) ( and g K) measured with 4dipyPC in DOPC as a function of water composition. Using Luzzatiœs approach36 we have determined the excess water volume fraction 0.42 and hence the average molecular cross-sectional area in excess water 72 Aé 2 for a molecular volume of DOPC of 1292 Aé 3.27 The samples contained 0.1 mol% 4dipyPC but this area is in agreement with measurements on pure DOPC by Gruner and co-workers.13 The same samples were used within two days of being made to measure g. At the lowest water composition we calculate that the molecular area has shrunk to 68 Aé 2 and g has declined by a factor of 2. volume of hydrocarbon moieties in —uid chains,35 we calculate that the pivotal surface for DOPE is located between C1 and C2 position on the hydrocarbon chain.Hence g should drop for all of our probes upon entering the inverse hexagonal phase and indeed this is what we –nd with g being 0.94 0.62 0.53 and 0.73 for 4- 6- 8- and 10dipyPC in DOPE at 25 °C. Furthermore the Fig. 8 ììLateral pressure pro–leœœ in the excess water L phase for three DOPC/DOPE compositions at 25 °C. a The excimer to monomer ratio for 4- 6- 8- and 10dipyPC are represented by bars of decreasing darkness. The error bars have been determined from the –t to the data in Fig. 5. Faraday Discuss. 1998 111 41»53 50 (Ö ),K L and respectively). The gradients dg/dT for 4- 6- 8- and 10dipyPC are Fig. 9 Response of dipyPC excimer formation to temperature for 80 mol% DOPE. g is plotted for 4- 6- 8- and 10dipyPC 0.051^0.003 0.040^0.001 0.035^0.001 and 0.045^0.003 respectively.calculations of Szleifer and co-workers10,11 show that the lateral pressure is always highest somewhere around the C3 position on the hydrocarbon chain and drops as one moves to the chain terminus. These observations are compelling evidence that although the pro–les of Fig. 8 do not give absolute values of n or z their qualitative appearance is consistent with the lateral pressure distribution of an oleoyl chain. Discussion Although the results we have reported only provide us with a qualitative picture of the lateral pressure pro–le in part of the chain region they tell us a number of interesting things. The –rst of these has already been stated and that is that although the net lateral chain pressure increases as we increase the monolayer torque tension it does so by decreasing the pressure near the head of the chains but increasing the pressure more rapidly beyond the cis double bond.This transfer of lateral pressure away from the region of the pivotal plane increases the torque tension more rapidly than simply increasing the lateral pressure uniformly along the chain. The increase in monolayer torque tension is brought about by the fact that adding DOPE to DOPC results in a reduction in the average molecular cross-sectional area. Clearly if the average area per molecule is reduced so is the exposure of hydrocarbon to water and hence the tension (negative pressure) at the polar/apolar interface will fall.Since the pressure in the chain region has increased this must mean that there has been a negative contribution to the lateral pressure in the headgroup region (the total lateral pressure must sum to zero). From this we deduce that phosphatidylethanolamine headgroups impart a negative pressure that is they exert an attractive force on phosphatidylcholine and -ethanolamine headgroups. We hypothesise that this attractive force is the result of direct hydrogen bond formation between headgroups. The pull between headgroups further increases the desire for the monolayer to bend. In the case of fully saturated chains Szleifer et al.10 –nd a single maximum in the lateral pressure. The position of this maximum relative to the length of the molecule moves towards the pivotal interface as the area is reduced.Since it is unlikely that we are collecting any data on the lateral pressure above the C4 position it is possible that something similar is occurring with the unsaturated oleoyl chains. Furthermore Szleifer and co-workersœ calculations indicate that as the 51 Faraday Discuss. 1998 111 41»53 area is reduced the rate of growth of the lateral pressure is most rapid near the chain ends. This is entirely consistent with our measurements. The feature which does not agree is that there is a drop in lateral pressure ; this is not seen in calculations on saturated chains. We do not believe that the drop we observe is an experimental artefact for example caused by the relatively bulky pyrene moieties having their motion hindered at shorter chainlengths.Given that g is approximately two times greater for 4dipyPC than any of the other probes it would seem it has little difficulty in forming excimers. Furthermore we –nd that g for 4- and 6dipyPC increases rapidly if we raise the temperature Fig. 9. This is in agreement with theoretical expectations that the pressure should scale with temperature as long as the cross-sectional area remains constant (for these small changes in temperature the area changes will not be as great as those imparted by changing the lipid composition). Further the fact that in this case g for 4- and 6dipyPC is seen to increase as we increase the torque tension contradicts the notion that some experimental complication is the cause of the decrease in g for 4- and 6dipyPC when we change the lipid composition.On balance we therefore conclude that the lateral pressure really does drop along some parts of the oleoyl chain as the cross-sectional area per chain is reduced and this remains to be explained theoretically. Conclusions This study has revealed a number of qualitative features of the changes in the lateral pressure pro–le brought about by an isothermal increase in the monolayer torque tension. The results indicate that DOPE drives mixed DOPC/DOPE monolayers into the inverse hexagonal phase by drawing headgroups into closer proximity. This in turn increases the total lateral pressure in the chain region and this is apparently brought about by the transfer of lateral pressure away from the region close to the pivotal surface to the opposite side of the cis double bond.Such subtle re-arrangements of the diÜerential lateral pressure are lost when we use the more familiar curvature elastic parameterisation of the torque tension. It is quite possible that two membranes may have identical torque tensions but distinctly diÜerent lateral pressure pro–les. Furthermore this work implies that the way in which the lateral pressure pro–le in such membranes changes under identical variations in the environmental conditions is likely to be diÜerent. It would be surprising if there were not some membrane proteins which were able to sense such subtle material diÜerences. The possible oversimpli–cation of correlating membrane protein properties with curvature elastic parameters makes the search for a lateral pressure probe worthwhile.However any further advances in our measurement and understanding of the micro-mechanics of the monolayer will require that we obtain quantitative measurements of the lateral pressure. To calibrate the probes is experimentally challenging. The rate of change of g with pressure could presumably be found by measurement upon application of an external pressure. However for this to be meaningful it would be necessary to do this in the L phase since in a solvent excimers can occur with chain a conformations which are simply not possible at an interface. The more vexed question is how one might –nd the value of g at zero lateral pressure. Put another way this would require one to measure g in the limit that the interfacial area per molecule tends to in–nity.Of course one cannot do this and nor could one extrapolate a series of measurements to zero pressure since the lateral pressure has yet to be established. It seems to us that a more realistic alternative is to calibrate measurements of g against a model system for which the lateral pressure has been determined from the statistical mechanics. This might also be used to calibrate the region of space probed by the excimer sensor. In the event that we were able to do such a thing there would still remain concerns about the perturbative eÜect of using a relatively bulky probe moiety such as pyrene on the lateral pressure. In this regard alternative smaller excimer forming moieties such as naphthalene might be better alternatives to pyrene.Finally it should be noted that we estimated a two fold change in the average chain lateral pressure between DOPC and DOPE. 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