DHPC Mixing in a Bicellar

Apr 6, 2001 - The degree of this mixed monolayer orientation at temperatures above the bicelle state is significantly lower. Near 60 °C a fraction of...
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Temperature Dependence of DMPC/DHPC Mixing in a Bicellar Solution and Its Structural Implications Edward Sternin,† David Nizza, and Klaus Gawrisch* Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, 12420 Parklawn Drive, Rockville, Maryland 20852 Received August 21, 2000. In Final Form: February 19, 2001 Bicelle-forming mixtures of short-chain 1,2-dicaproyl-sn-glycero-3-phosphocholine (DHPC) and longchain 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) exhibit complicated phase behavior. We studied chain-melting and morphological phase changes in mixtures of DMPC/DHPC of 4.5:1 molar ratio in the temperature range of 0-60 °C. The phase state of both lipids and the alignment of lipid monolayers in the magnetic field were determined by solid-state 2H and 31P NMR. DMPC and DHPC mix in a micellar state at 0-10 °C but separate upon heating to 15 °C into gel-phase DMPC bilayers and DHPC micelles. Near the gel-to-liquid-crystalline phase transition of DMPC, at 24 °C, the DHPC spectra become anisotropic suggesting arrangement of DHPC in a mixed DMPC/DHPC phase. DMPC in this structure possesses the chain order typical of liquid crystalline bilayers, but DHPC chain order remains much lower, with lipid aggregates oriented mostly at random. In the bicelle phase, at 32-36 °C, highly anisotropic lipid particles with their DMPC monolayer normal oriented perpendicular to the magnetic field are formed. The angular distribution function of bilayer normals has significant mosaic spread and tails with surprisingly high intensity that is caused by the monolayers that are oriented at random. In the bicelle phase, magic angle spinning nuclear Overhauser effect 1H NMR spectrometry revealed intensive intermolecular cross-relaxation among DMPC and among DHPC molecules but no detectable magnetization exchange between DMPC and DHPC, confirming that DHPC and DMPC experience limited physical contact. Above 36 °C, chain order of DMPC decreased, in particular for the bonds near the terminal methyl groups, while order parameters of DHPC increased, both indicative for DMPC/DHPC mixing in the same monolayer. The degree of this mixed monolayer orientation at temperatures above the bicelle state is significantly lower. Near 60 °C a fraction of DHPC converts to a micellar phase that also dissolves a trace of DMPC. Thus, a highly aligned bicelle phase exists only in a narrow range of temperatures. Complex orientational distributions arise at other temperatures, driven by lipid mixing and structural phase changes.

Introduction Despite the widespread use of 1,2-dimyristoyl-snglycero-3-phosphocholine (DMPC)/1,2-dicaproyl-sn-glycero-3-phosphocholine (DHPC) bicelles for high-resolution NMR investigation of protein structure,1-4 little is known about temperature-induced phase transitions and changes of lipid arrangement in mixed DMPC/DHPC/water liquidcrystalline phases. Groundwork NMR spectroscopy,5,6 a limited line shape analysis through the spectral first moments,7 and the response elicited from the phospholipid headgroups by the surface charges,8 are all consistent with a bicellar phase made up of discoidal patches of longchain phospholipid bilayer with short-chain phospholipid “end-caps” or “rims” sealing the bilayer from the aqueous medium.6,9 However, a closer examination of published 31P NMR spectra5,10 reveals anisotropic broadening. It was reported that the poor resolution may result from poor alignment of bicelles in the magnetic field or from some † Permanent address: Brock University, St. Catharines, ON L2S 3A1, Canada.

(1) Sanders, C.; Hare, B.; Howard, K.; Prestegard, J. Prog. NMR Spectrosc. 1994, 26, 421. (2) Vold, R.; Prosser, R. Deese, A. J. Biomol. NMR 1997, 9, 329. (3) Tjandra, N.; Bax, A. Science 1997, 278, 1111. (4) Bax, A.; Tjandra, N. J. Biomol. NMR 1997, 10, 289. (5) Sanders, C.; Schwonek, J. Biochemistry 1992, 31, 8898. (6) Sanders, C.; Landis, G. Biochemistry 1995, 34, 4030. (7) Picard, F.; Paquet, M.; Levesque, J.; Belanger, A.; Auger, M. Biophys. J. 1999, 77, 888. (8) Crowell, K.; Macdonald, P. Biochim. Biophys. Acta, Biomembranes 1999, 1416, 21. (9) Vold, R.; Prosser, R. J. Magn. Reson., Ser. B 1996, 113, 267. (10) Losonczi, J.; Prestegard, J. J. Biomol. NMR 1998, 12, 447.

10.1021/la001199w

distribution in bicelle composition and size.2 It is also possible that bicelles are not strictly a dispersion of uniform-size disks but instead form some interconnected structure.8,11-13 Our goal was to identify the lipid phases that are formed when a DMPC/DHPC bicelle mixture is heated from 0 to 60 °C, and to determine the degree of lipid monolayer alignment in these phases. To follow both lipids individually, we prepared mixtures of DMPC/DHPC (molar ratio q ) 4.5) with only one of the two lipids deuterated at a time. Solid-state 31P and 2H NMR experiments were conducted over the range of 0-60 °C at temperature increments of 5 °C or less. Every sample was investigated according to the same temperature protocol. NMR equipment that was especially designed for investigations of lipid bilayers and NMR echo sequences that minimize signal distortions from probe ringing were employed14,15 to obtain spectra that are free of baseline artifacts and phase errors. Using well-known techniques to identify phase states of lipids from solid-state 31P and 2H NMR data,16,17 we tentatively assigned structural phases to individual lipid components. Assignment is mostly based on the analysis (11) Prost, J.; Rondelez, F. Nature 1991, 350 (Supplement), 11. (12) Katsaras, J.; Donaberger, R.; Swainson, I.; Tennant, D.; Tun, Z.; Vold, R.; Prosser, R. Phys. Rev. Lett. 1997, 78, 899. (13) Sanders, C.; Prosser, R. Structure 1998, 6, 1227. (14) Davis, J. H.; Jeffrey, K. R.; Bloom, M.; Valic, M. I.; Higgs, T. P. Chem. Phys. Lett. 1976, 42, 390. (15) Rance, M.; Byrd, R. A. J. Magn. Reson. 1983, 52, 221. (16) Seelig, J. Q. Rev. Biophys. 1977, 10, 353. (17) Seelig, J.; Seelig, A. Q. Rev. Biophys. 1980, 13, 19.

This article not subject to U.S. Copyright. Published 2001 by the American Chemical Society Published on Web 04/06/2001

Structure of DMPC/DHPC Bicelles

of the 2H NMR spectra of perdeuterated lipid hydrocarbon chains of DMPC and DHPC. We also followed the phase transitions by 31P NMR. Although interpretation of 31P NMR results is difficult due to signal superposition, the spectra proved invaluable for comparison of phase states between lipid mixtures of deuterated DMPC or deuterated DHPC, since deuteration resulted in lipid-specific shifts of phase transition temperatures. Spectra were then analyzed by dePakeing, a numerical procedure that converts the experimentally measured spectra of randomly oriented structures into the corresponding spectra of perfectly aligned monolayers.18-21 Recently developed powerful regularization techniques enable us to dePake spectra of partially aligned lyotropic liquid crystals.22 When applied to high-fidelity NMR spectra, these techniques allow us to simultaneously extract both the underlying orientational distribution function of the monolayer normals with respect to the external magnetic field and the average motional order parameters of individual lipid segments. The former is a function of the structural organization of the sample, while the latter is a measure of the efficiency of rapid reorientational motions of molecular segments. Mixing of DHPC and DMPC in the bicelle state at 3236 °C was also investigated by 1H magic angle spinning NOESY spectroscopy. As we have shown earlier,23 NOESY cross-relaxation within liquid-crystalline lipid-water dispersions is predominantly of intermolecular origin. The experiments allowed judgment about the distribution of DMPC and DHPC in the bicelle phase. Materials and Methods Bicelle Preparation. The lipids 1,2-dimyristoyl-sn-glycero3-phosphocholine (DMPC), 1,2-dicaproyl-sn-glycero-3-phosphocholine (DHPC), and their deuterated analogues, 1,2-dimyristoyld54-sn-glycero-3-phosphocholine (d54-DMPC), 1,2-dicaproyl-d22sn-glycero-3-phosphocholine (d22-DHPC) were purchased from Avanti Polar Lipids (Alabaster, AL) as dry powders and dehydrated under vacuum for several hours prior to use. DHPC was dried under vacuum in the presence of phosphorus pentoxide for 24 h. Two slightly different sample preparation methods were used. For the magic-angle spinning (MAS) experiments, dry lipids were added in the right proportions to achieve the desired q, the molecular ratio of the long-chain (DMPC) to short-chain (DHPC) lipids, under dry nitrogen gas. A solution of 0.05% NaN3 in D2O was added by volume and verified by weight afterward, the sample was sealed under Ar, and several freeze-thaw cycles (typically, five to eight) were performed, with gentle vortexing as the sample was melting. The samples were then equilibrated at room temperature for 12 h and transferred into the MAS rotor. Additional MAS samples and all subsequent samples for the solid-state 31P and 2H NMR experiments were made using stock solutions of DHPC or d22-DHPC, prepared under dry nitrogen gas. An appropriate amount of dry DMPC powder was weighed into a 250-µL glass vial with ground glass stopper. DHPC solution and, subsequently, deuterium-depleted water containing 150 mM NaCl were added to achieve the desired q and the total lipid concentration of 25 w/v %.8 Each step was verified gravimetrically. The vial was sealed and equilibrated over 24 h by performing thermal equilibration at 4 °C and then 40 °C. This was repeated 10 times in a 12-h period, concluded by a final equilibration at 40 °C followed by cooling to room temperature. Both methods of (18) Bloom, M.; Davis, J. H.; MacKay, A. L. Chem. Phys. Lett. 1981, 80, 198. (19) Sternin, E.; Bloom, M.; MacKay, A. L. J. Magn. Reson. 1983, 55, 274. (20) Whittall, K.; Sternin, E.; Bloom, M.; MacKay, A. J. Magn. Reson. 1989, 84, 64. (21) Scha¨fer, H.; Ma¨dler, B.; Volke, F. J. Magn. Reson., Ser. A 1995, 116, 145. (22) Scha¨fer, H.; Ma¨dler, B.; Sternin, E. Biophys. J. 1998, 74, 1007. (23) Huster, D.; Arnold, K.; Gawrisch, K. J. Phys. Chem. B 1999, 103, 243.

Langmuir, Vol. 17, No. 9, 2001 2611 preparation yielded transparent viscous liquid samples of uniform consistency, and the MAS spectra from the two methods of sample preparation were indistinguishable. NMR Spectroscopy. Solid-state NMR experiments were performed on a Bruker DMX300 wide-bore spectrometer at 46.07 MHz for 2H and at 121.5 MHz for 31P; MAS 1H experiments were performed at 500.13 MHz and 10 kHz spinning speed on a Bruker DMX500 wide-bore spectrometer, using a double gas bearing MAS probe-head with 4 mm rotors. The 12-µL inserts with screwtype seals assured no evaporation and thus no change in water content over the course of the experiments. The temperature control to 0.1 °C was achieved using a Bruker VT controller, with air passing through a cooling bath for temperatures below 25 °C. Temperature calibration within the MAS rotor was achieved by following known phase transition temperatures of pure lipid samples. Every sample was equilibrated for at least 20 min before collecting data, at each temperature reported here. To minimize thermal hysteresis, we always acquired the spectra from lower to higher temperatures, and re-equilibrated the samples at 4 °C every time the sample temperature had to be lowered. In this way, all samples had a consistent thermal history. Nuclear Overhauser enhancement (NOESY) data sets (256 × 1024 2D) were acquired with 16 scans per t1 increment, a mixing time of τmix ) 300 ms, and a recycle delay of 6 s, using the methods published previously.23-25 Shorter mixing times were also examined to verify that 300 ms was optimal for NOE transfer. Volumes of partially superimposed resonances in the 2D spectra were determined by deconvolution assuming that resonances have Gaussian line shape. Peak intensities, as well as positions and line widths in both dimensions were optimized by software utilizing the Levenberg-Marquardt algorithm.26 Numerical Methods. Numerical techniques allow highly detailed data analysis, but for that to be successful extra care must be taken to obtain high-fidelity 1D spectra. We were careful to avoid zero filling and running-average digital filtering, routinely used in commercial software. Analogue filters set to match the digitization bandwidth may display considerable nonlinearity well into the spectral regions of interest; we used the settings that assured a flat response curve over the entire measured spectrum. The quadrupolar echo pulse sequence14 was used to acquire data without dead-time delays of the digitizer; prior to Fourier transform, the time-domain signal was carefully recalculated to yield a point at the top of the echo using a fifthorder polynomial interpolation algorithm,27 incorporated into a custom data processing software package.28 Traditional dePakeing18-21 deals well with single-phase systems that exhibit little or none of the alignment effects of bicelles. Recent developments have made it possible to extract true anisotropy distributions even in the presence of nonrandom orientational distributions within the powder sample that may arise under the influence of the magnetic field on the model membrane systems with anisotropic magnetic susceptibility.22 In our case, dePakeing was performed using the Tikhonov regularization algorithm

Ψ ) ||S(ω) -

∫g(x)k(ω,x) dx|| + λT[g(x)]

(1)

which minimizes a quadratic misfit between the input spectrum and the one recalculated from the dePaked spectrum.21 Here, S(ω) is the experimentally measured spectrum, g(x) is the distribution function of anisotropies (such as the quadrupolar order parameters) present in the sample and is thus the desired result of the calculation together with the orientational distribution parameter(s), k(ω,x) is the kernel function that represents the line shape one obtains for each anisotropy x, T[g(x)] is the Tikhonov regularization term, and λ controls the balance between (24) Holte, L. L.; Gawrisch, K. Biochemistry 1997, 36, 4669. (25) Huster, D.; Gawrisch, K. J. Am. Chem. Soc. 1999, 121, 1992. (26) Press: W.; Flannery, B.; Teukolsky, S.; Vetterling, W. Numerical recipes. The art of scientific computing; Cambridge University Press: Cambridge, Great Britain, 1989. (27) Davis, J. H. In Isotopes in the Physical and Biomedical Sciences; Buncel, E., Jones, J. R., Eds.; Elsevier: Amsterdam, 1991; Vol. 2; pp 99-157. (28) Sternin, E. danssData Analysis of NMR Spectra. Available at http://www.physics.brocku.ca/faculty/sternin.html, 1999.

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compatibility with the data and the regularizing effects of T[g(x)]. We used a recently developed numerical procedure that allows simultaneous extraction of the orientational distribution function in a partially aligned system as well as the dePaked spectrum itself.22 The three different model forms of the orientational distribution function used in that work (ellipsoidal, Boltzmann, and Legendre) represent different ways of introducing physically meaningful trial functions which enable one to consider deviations from the random orientational distribution of a perfect powder. Each of the trial functions is parametrized by a single parameter that yields the extent of orientational enhancement in the sample. As the misfit is minimized, the best value of this parameter is determined for each trial model; the lowest minimum represents the best-fitting model. In the case of bicelles, the added complication of possible coexistence of multiple structural phases required the introduction of a generalization of the method. The shape of low-intensity tails of the observed NMR spectra was not consistent with a single partially aligned phase. Thus, in addition to the aligned lipid fraction, a second component oriented at random and represented by a contribution to p(θ) that is proportional to sin(θ) was included. The full trial orientational distribution function was

p(θ) ∝ fE sin θ[1 - (1 - κE) cos2 θ]-2 + (1 - fE) sin θ (2) where the two parameters of the model were fE, the relative fraction of the ellipsoidal phase, and κE, the extent of the ellipsoidal distortion that has a simple geometrical meaning of the square of the ratio of the two semiaxes of an ellipsoid. We have also examined other models where the ellipsoidal part of p(θ) was replaced with that appropriate for the Boltzmann, ∝sin θ exp[κB cos2 θ], or Legendre, ∝sin θ[1 + κL cos2 θ], models. In all cases, the results were indistinguishable from those for the ellipsoidal model, with misfit minima of similar depth, and thus only the results obtained using eq 2 are reported here. In all instances, convergence was below two standard deviations of the distribution of the random noise, as measured in the baseline regions of the spectrum. In addition, fits were rejected if there was any systematic misfit appearing below that level. Further details of the methods are presented in a separate publication.29 To assist in the interpretation of the dePaked spectra as anisotropy distributions, they are integrated and the resulting integral divided into an appropriate number of intervals, one for each carbon position along the chain, and the center of each interval is denoted by a tick mark.30,31 The centermost peaks are known to belong to the terminal methyl groups on each chain, and there are known relaxation rate differences between those and all other carbon positions. Since this is expected to affect the measured intensities in these peaks, they are excluded from this integration, as their appropriate quadrupolar splittings could be assigned by hand, directly from the resolved position of the peak of each line. Where this is not possible, the integration limits are extended to zero frequency, but the relative weight of the methyl group (innermost) peaks is assumed to be 1.5 that of the other peaks. Also, the carbon nearest to the glycerol in d54-DMPC and likely to correspond to the largest quadrupolar splitting is only partially deuterated, as determined by MAS NMR experiments (not shown). Hence the weight of the outermost peaks in DMPC order parameter distributions is set to 0.5 that of the other peaks. In addition, although both sn-1 and sn-2 chains are identical on each molecule, there are known significant differences in order parameters between the two chains; hence, the integration range is divided into the number of intervals which is twice the number of carbons in one chain, i.e., 8 for d22-DHPC and 24 for d54-DMPC. This simple-minded approach yields excellent agreement with those peaks that are resolved individually; it is essentially the only way to assign quadrupolar splittings, and, therefore, the order parameters, in those other regions where peaks overlap. (29) Sternin, E.; Scha¨fer, H.; Polozov, I.; Gawrisch, K. J. Magn. Reson., in press. (30) Sternin, E.; Fine, B.; Bloom, M.; Tilcock, C.; Wong, K.; Cullis, P. Biophys. J. 1988, 54, 689. (31) Lafleur, M.; Fine, B.; Sternin, E.; Cullis, P. R.; Bloom, M. Biophys. J. 1989, 56, 1037.

Sternin et al. We only present the results of dePakeing of the 2H NMR spectra. Clearly, 31P NMR spectra could also be dePaked; however since both constituent lipids have the same choline headgroup, the results could not be made lipid-specific. In addition, for highly oriented samples the spectra exhibit long low-intensity tails which make a precise determination of the zero of the first moment difficult in the presence of noise. 2H NMR spectra do not present either of these difficulties.

Results (A) H and P NMR Spectra Reveal a Complex Phase Diagram. (1) Mixed-Lipid Micelles between 0 and 10 °C. Short-chain, detergent-like phospholipids such as DHPC are known to dissolve in the aqueous medium, whether as single molecules or as small micellar structures. DHPC molecules are capable of dissolving DMPC molecules and maintaining them in a micellar state below the gel-to-liquid-crystalline (chain-melting) phase transition of DMPC. With this possibility in mind, we examine the NMR spectra of Figure 1 as a function of temperature. At the lowest temperature shown, spectra consist of narrow isotropic peaks typical of rapidly reorienting molecules in small micelles. This is not what is normally seen in DMPC at these temperatures and suggests that significant mixing of DMPC and detergentlike DHPC is taking place. (2) DMPC Precipitates into Gel State Bilayers at 15 °C. At around 15 °C, the broad gel phase patterns emerge in both 31P and 2H DMPC spectra, yet the 2H DHPC spectrum broadens insignificantly. This suggests a phase separation into a DMPC-rich phase, which begins to resemble the multilamellar vesicles typical of the pure DMPC samples at these temperatures, and an almost pure DHPC phase of relatively small vesicles or micelles. (3) Partial Mixing of DMPC and DHPC in the 2030 °C Range. Near 24 °C the DMPC converts to a liquid crystalline lamellar phase as confirmed by the reduction of 31P anisotropy of chemical shift and the change in appearance of DMPC 2H hydrocarbon chain spectra. The 2 H spectrum of DHPC hydrocarbon chains shows indications of a weak anisotropy in molecular motions. Furthermore, the narrow resonance in the 31P spectrum, which at this temperature is almost exclusively caused by DHPC, shifts slightly to higher field indicating the presence of a small anisotropy of chemical shift. DHPC must have gained this anisotropy in molecular motions by interaction with ordered DMPC. However, order parameters of DHPC remain very low, indicating that contact is limited. (4) Sudden Alignment Accompanies Bicelle Phase, 32-36 °C. Over the temperature range from 30 to 32 °C the system undergoes a structural transition that is characterized by the appearance of significant alignment of DMPC lipid monolayers with their normal oriented perpendicular to the magnetic field. Compared to spectra at lower temperature, there is a modest reduction of DMPC chain order and a significant increase in DHPC chain order. The DHPC order increase is confirmed by the highfield shift of the weaker resonance in the 31P spectrum. Despite the increase, DHPC order parameters remain significantly lower, indicating more isotropic motions. The nature of the spectra is typical of axially symmetric motions that are rapid on the NMR time scale; thus the lower order parameter values are most likely due to additional quadrupolar interaction-modulating motions that a DHPC molecule participates in, as compared to a DMPC molecule. A higher local curvature in the regions rich in DHPC, as attributed to the “rims” of the bicelles, is consistent with such spectra. Spectra maintain their appearance over a narrow temperature interval from 32 to 36 °C only. 2

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Structure of DMPC/DHPC Bicelles

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Figure 1. NMR spectra of DHPC/DMPC mixture. 31P and 2H spectra from two samples, a q ) 4.4 mixture of d54-DMPC/DHPC and a q ) 4.55 mixture of DMPC/d22-DHPC reveal several structural and chain-melting transitions. Because of the influence of deuteration on phase transition temperatures, the temperatures at which the measurements were made are slightly different in the two samples. For a precise comparison, the temperatures for the DMPC/d22-DHPC sample (right-hand column) were adjusted until the 31P spectra exhibited lineshapes identical with those of the d54-DMPC/DHPC; only the d54-DMPC/DHPC set is shown (left-hand column). Thus the two sets of 2H spectra represent a comparison of the molecular order within DMPC (center) and within DHPC (right-hand side) at approximately the same points in the phase diagram. Note the horizontal scale in the two sets of 2H data.

(5) Remixing of DMPC and DHPC within Two Separate Structures above 36 °C. At 37 °C, two lipid phases emerge that change continuously in lipid order and composition with increasing temperature. The 2H hydrocarbon chain spectra of DMPC show some redistribution of chain order with more emphasis on lower order parameters. According to the 2H spectra of DHPC, one phase decreases in lipid order and volume with increasing temperature, while the second phase takes up increasing amounts of DHPC with chain order similar to that of DMPC. Near 60 °C a small fraction of DHPC forms an isotropic, presumably micellar, phase that also dissolves a trace of DMPC. This phase disappears completely when the sample is cooled to 0 °C. Therefore, the isotropic signal is not a result of lipid chemical degradation. The dramatic rearrangement at temperature above 37 °C with coexistence of phases is confirmed by the 31P spectra. Intensity of peaks in the 31P spectra can no longer be assigned to a particular lipid. (B) 1H NOESY Confirms Short- and Long-Chain Lipid Separation in Bicelle Phase. In previous work, it has been established that intermolecular interaction in lipid mixtures is responsible for cross-peaks in the NOESY spectra;23,25,32 the intensity of such peaks reaches its maximum at τmix = 500 ms for a typical model membrane (32) Feller, S.; Huster, D.; Gawrisch, K. J. Am. Chem. Soc. 1999, 121, 8963.

involving DMPC. For a homogeneous mixture of q ) 3.1, one would expect cross-peaks between DMPC and DHPC approximately three times above the threshold concentration of about 10% DHPC in DMPC that can be detected using these methods. However, none is seen in the spectrum of Figure 2, taken at 35 °C which corresponds to the bicellar phase. Thus on the time scale of the experiment, the two lipid species in the bicellar mixture do not appear to have significant contact with each other. Such lateral separation of lipid species is an unusual feature for a liquid crystalline phase of highly mobile lipid molecules; and it is in good agreement with the postulated bicelle shape of discoidal bilayer patches of DMPC surrounded by a “rim” of DHPC. One must add that other structures that exhibit the same characteristic separation of constituent lipids are possible and need to be considered. Outside of the bicelle phase, multiple mixed DMPC/ DHPC environments exist within the sample. The chemical shifts of the hydrocarbon chain terminal methyl groups of each of the lipid species differ within these mixed environments, and the observed separate peaks can no longer be uniquely assigned to DMPC or DHPC. Thus a direct 2D NOESY confirmation of such mixing, similar to Figure 2 but containing cross-peaks, is not possible. (C) 2H NMR Measures Motionally Averaged Order Parameters. A more informative representation of the spectroscopic information of Figure 1 is in terms of the so-called dePaked spectra, those that correspond to a

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Figure 2. 2D 1H NOESY spectrum of DHPC/DMPC mixture. A q ) 3.1 ratio mixture, with 75% D2O by weight, at 35 °C and 10 kHz spinning rate. The lines indicate the connectivity pattern between the terminal methyls of both lipids (0.9 and 0.95 ppm), the third carbon (CH2) along each chain (1.63-166 ppm), and the bulk of (CH2) groups in the middle of the phospholipid chains (1.31-1.35 ppm). Solid lines correspond to DHPC, and dashed lines correspond to DMPC connectivity patterns. The assignment is based on the similar spectra of mixtures where one of the lipid components was deuterated, not shown here. Strong intermolecular cross-peaks between identical lipid molecules are seen, yet even with peak deconvolution we are unable to detect any cross-peaks between DHPC and DMPC molecules.

perfectly aligned bilayer phase with its bilayer normal at a certain fixed angle θ with respect to the magnetic field. Conventionally, dePaked spectra are displayed as appropriate for θ ) 0. The integral intensity in such spectra directly represents the distribution function of anisotropies present in the system, such as the chemical shift anisotropies (for 31P NMR) or quadrupolar order parameters (for 2H NMR). Bicelles are aligned by the magnetic field, and so their spectra in Figure 1 (32 °C for d54-DMPC:DHPC, and 36 °C for DMPC:d22-DHPC) are easier to analyze; other spectra are from mixed and, at best, only partially aligned phases and require more extensive numerical processing. The nonrandomness of the orientational distribution in DHPC/DMPC mixtures is particularly difficult to deal with, as magnetic orientational effects overlap with the effects due to the coexistence of multiple structural arrangements. Fortunately, the orientational distribution function appears to be fairly well approximated by a combination of a random (spherical) contribution and that from an ellipsoid of rotation with its long axis along the external magnetic field. The numerical analysis using the methods described above (see Materials and Methods) allows us to extract the dePaked spectra and, consequently, the anisotropy distribution functions. The reported results are not sensitive to the details of the model used to parametrize the orientational distribution within the sample (two other trial models, using Boltzmann or Legendre distribution functions, yield very similar results), and thus we report the dePaked spectra of Figure 3 with confidence. We concentrate on the bicellar and the adjacent phases and, therefore, show here only some of the anisotropy distributions extracted from the spectra in the upper-right section of Figure 1. From data analysis it is obvious that the orientational distribution function of lipid bilayers changes dramatically with temperature. Lipid bilayers at temperatures below

Sternin et al.

32 °C show a small preference for orienting the bilayer normal perpendicular to the magnetic field. The observed distribution functions are consistent with a slightly elongated (κE ) 1.5) ellipsoidal distribution. Preferential orientation improves drastically in the bicelle phase (3236 °C). There the distribution is well approximated by 87% ellipsoidal, highly elongated (κE ) 35) distribution in combination with a 13% random contribution. At temperatures above the bicelle phase the degree of alignment declined somewhat, with 75% ellipsoidal (κE ) 15) plus 25% random contribution. Unfortunately, the complexity of multiple phases in coexistence throughout most of the examined temperature range prevents us from making an unambiguous determination of the order parameter profile, S(n), as a function of the carbon number, n, along the fatty acid chain. However, the dePaked spectra act as a useful aid in establishing the phase behavior of the system. For example, at the temperature of 21 °C the second innermost pair of lines in the spectrum of d54-DMPC appears to have lower than usual relative weight of about 0.6. This is consistent with about one-third of DMPC being part of another structure. Spectral resolution of other peaks is insufficient to draw further conclusions. Furthermore, the order parameter distribution functions of DMPC in the bicelle phase (32 °C) and at higher temperatures (58 °C) differ substantially. The bicelle spectrum of DMPC is typical for an order parameter distribution with an order parameter plateau for half of the chain near the glycerol and a rapid decline in order toward the terminal methyl group of the chain. At higher temperatures order of the fraction of chain segments near the terminal methyl groups is reduced reflecting the decline in myristic acid order when mixed with short-chain caproic acid. The substantial order differences between DMPC and DHPC in the bicelle phase confirm that both lipids are segregated. The order increase seen in the dePaked spectra of DHPC in combination with the change in the order parameter distribution of DMPC confirms remixing of both lipids at higher temperatures. Toward a Partial Phase Diagram The schematic diagram of Figure 4 summarizes the complex structural phase behavior of the DHPC/DMPC mixtures as seen through the temperature-induced changes in their NMR spectra. The narrow isotropic spectra at the bottom of Figure 1 imply fairly small, likely micellar, structures shown in Figure 4a; since pure DMPC at these temperatures is in the gel state, it is necessary that at least some mixing of DMPC and DHPC occurs. The resulting phase is probably similar to mixed micelles observed in lipid-detergent mixtures;33,34 its unusual appearance at a lower temperature than the smectic phase has been confirmed before using neutron scattering.12 As the temperature is raised, the two observed 31P chemical shifts correspond to two very different structures coexisting, and the 2H spectra suggest demixing of DHPC and DMPC, with DMPC being in an essentially lamellar phase as shown in Figure 4b; note, for example, a “plateau” region typical of the bilayer phase observed in the distribution of tick marks for DMPC at the two lower temperatures in Figure 3. As the chains melt, the two structural phases are in rapid exchange, as indicated by the arrow in Figure 4c; a significant broadening of the 2H spectra of d22-DHPC (33) Kessi, J.; Poiree, J.; Wehrli, E.; Bachofen, R.; Semenza, G.; Hauser, H. Biochemistry 1994, 33, 10825. (34) Kraghhansen, U.; Lemaire, M.; Noel, J.; Gulikkrzywitcki, T.; Moller, J. Biochemistry 1993, 32, 1648, 1648.

Structure of DMPC/DHPC Bicelles

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Figure 3. Order parameter distributions as seen in the dePaked spectra of DHPC/DMPC mixtures. Mixed-model dePakeing using the Tikhonov regularization technique22 of the spectra in the upper-right section of Figure 1 yields these “dePaked” spectra over a fairly broad range of orientational distribution parameters. All orientational distributions indicate varying degrees of alignment, and the convergence in the upper three rows in Figure 1 can only be achieved if, in addition, a combination of a partially aligned and a random contribution is assumed to be present. The relative weight of the two contributions and the degree of alignment are the two nonlinear fit parameters. The fit proceeds until the results are indistinguishable from the spectra in Figure 1, to within 1 standard deviation of the noise. The ticks shown below each dePaked spectrum represent the observed order parameter distribution, as extracted by integrating the dePaked spectra and dividing the integral into an appropriate number of intervals. The reported fE and kE correspond to the best fit values 2%. See text for further details.

the bicellar phase (Figure 4d) is characterized by lateral separation of DHPC and DMPC. This is confirmed by the absence of the cross-peaks between the DHPC and DMPC in 2D NOESY spectra (Figure 2). At the same time, the system undergoes a dramatic orientational ordering. It is not clear whether “flat DMPC disks with DHPC rims” or “DMPC vesicles with DHPC pores” is a better shorthand description of the geometry of this phase. The orientational ordering is partially maintained at still higher temperatures, where a mixed-lipid isotropic phase emerges and coexists with the aligned lamellar (bicellar?) structures (Figure 4e); note the broad isotropic line making its appearance in the top distributions shown for both DMPC (on the left) and DHPC (on the right) of Figure 3. Existence of two separate resonances suggests that the lifetime of lipids in these structures is 10-3 s or longer. Both the width of the central peak and the observed quadrupolar splittings of the outermost lines in both DMPC and DHPC approach each other, suggesting mixing of the two phospholipids within each of the two phases. Concluding Remarks Figure 4. Diagrammatic summary of structural phases seen in DHPC/DMPC mixtures. The order (top-to-bottom) corresponds to that of the spectra in Figures 1 and 3; see text for a detailed discussion. Magnetic alignment is only observed for the structures in parts d and, partially, in e. The headgroups of DHPC are represented by filled circles for visual clarity only, as the PC headgroup is the same in both DHPC and DMPC. The temperature ranges shown correspond to the nondeuterated DHPC/DMPC mixtures and are only approximate; deuteration of one of the components may shift the temperatures by several degrees.

means that a rearrangement of mixed DMPC/DHPC structures occurs. As the temperature is raised further,

The primary purpose of this study was to investigate structural changes that occur in bicelle mixtures as a function of temperature. This was achieved by 2H-labeling DHPC or DMPC individually. We observed chain-melting and morphological phase changes with phase coexistence at most temperatures above and below the rather narrow temperature range of the bicelle phase, 32-36 °C. An essential part of these experiments was our ability to measure both 31P and 2H spectra without changing NMR probe heads, while maintaining the sample at a given temperature. Only by matching the 31P spectra between the three bicellar samples (DMPC/DHPC, d54-DMPC/ DHPC, and DMPC/d22-DHPC) were we able to remove

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the ambiguity of the state of the sample in the complicated phase diagram, as various transitions occur at slightly different temperatures in the two samples. 31P NMR proved a highly efficient tool for monitoring the thermodynamic state of the sample. It should also be noted that all of the spectra presented are reproducible as long as care is taken to maintain a consistent thermal history of the sample (see in Materials and Methods, above). DHPC/DMPC mixtures are aligned by an external magnetic field to a varying degree, depending on the exact structural state of the system. A traditional model of flat bilayer patches of the long-chain phospholipid surrounded by a “rim” of the short-chain one, all oriented with bicelle normals perpendicular to the external magnetic field, is only consistent with our spectra over a very narrow range of temperatures, 32-36 °C. Even there, it is impossible to exclude other possible orientational models, such as highly elongated tubular structures that can be approximately thought of as ellipsoids of rotation with the ratio of the short and long semiaxes of about 6. Away from these “optimum” temperatures, we observed dramatic changes in the orientational distribution functions reported by the algorithm; we have strong indications of highly oriented fractions in coexistence with randomly oriented ones. Further work is required to provide a better understanding of what orientational models are appropriate to these complex mixed phases; however, the shape of the dePaked spectra appears to be insensitive to the exact details of the model. Thus order parameter distribution and its changes through various structural rearrangements of the system can be reported here independently of the changes in the orientational distribution function.

Sternin et al.

It appears that outside of a narrow temperature range, 32-36 °C, what is thought of by most researchers as slightly imperfect bicelles are, in fact, multiple structural phases coexisting. The lipid bilayers of these phases are only partially aligned by the magnetic field. It is an unfortunate coincidence that since the preferred orientation corresponds to θ ) 90°, the already prominent peaks of the powder spectra are enhanced. Even a mild degree or partial magnetic alignment (for example, as in an ellipsoid of rotation with the ratio of long to short semiaxes of only about 3-4) produces a visually dramatic “enhancement” of the observed powder spectra in just the right way to imitate a highly aligned phase. However, with careful attention to the exact line shape of the (highfidelity!) spectra, these effects of partial magnetic alignment can be quantified and, in fact, used to characterize the exact structural makeup of the system. This may be a necessary, albeit laborious and computationally intensive step in understanding the data from peptides and proteins incorporated into bicelles. Acknowledgment. We are grateful to Dr. H. Scha¨fer for the use of his Tikhonov regularization software and for helpful discussions. E.S. wishes to acknowledge the financial support of the Natural Sciences and Engineering Research Council of Canada and the intramural support from the Laboratory of Membrane Biochemistry and Biophysics, NIAAA, NIH, during the sabbatical stay. LA001199W