DHPC Bicelle Formation and

May 1, 2012 - When detergent meets bilayer: Birth and coming of age of lipid bicelles. Ulrich H.N. Dürr , Ronald Soong , Ayyalusamy Ramamoorthy. Prog...
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Effect of Divalent Cations on DMPC/DHPC Bicelle Formation and Alignment Amanda J. Brindley† and Rachel W. Martin*,†,‡ †

Department of Chemistry and ‡Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, California 92697, United States S Supporting Information *

ABSTRACT: Many important classes of biomolecules require divalent cations for optimal activity, making these ions essential for biologically relevant structural studies. Bicelle mixtures composed of short-chain and long-chain lipids are often used in solution- and solid-state NMR structure determination; however, the phase diagrams of these useful orienting media and membrane mimetics are sensitive to other solution components. Therefore, we have investigated the effect of varying concentrations of four divalent cations, Ca2+, Mg2+, Zn2+, and Cd2+, on cholesterol sulfate-stabilized DMPC/DHPC bicelles. We found that low concentrations of all the divalent ions are tolerated with minimal perturbation. At higher concentrations Zn2+ and Cd2+ disrupt the magnetically aligned phase while Ca2+ and Mg2+ produce more strongly oriented phases. This result indicates that divalent cations are not only required to maintain the biological activity of proteins and nucleic acids; they may also be used to manipulate the behavior of the magnetically aligned phase.

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negatively affect the catalytic activity of such MMPs.25 Bilayer structures with a lower radius of curvature are sometimes required to ensure native conformations26,27 and can be produced using lipids alone or with a “belt” protein providing a template for uniform nanodisks that can be used for characterization of membrane proteins.28,29 In the case of solution-state NMR on soluble proteins, the bilayer environment is incidental; bicelle mixtures are used to induce weak alignment, enabling measurements of residual dipolar couplings30−32 and chemical shift offsets due to CSA,33−35 from which distance and orientational constraints, respectively, can be extracted. Bicelle mixtures made from a mixture of short- and longchain phospholipids self-assemble into ordered liquid crystalline domains containing bilayer regions36 that can allow insertion of membrane proteins.37 The morphology of a bicelle preparation depends on the q value (ratio of long-chain to short-chain lipids) and temperature, such that variations in lipid composition and concentration can be used to produce a range of phases from small, fast-tumbling bicelles to strongly aligned perforated lamellae38 or wormlike micelles.39 The most commonly used bicelle mixture is DMPC/DHPC, which is fairly robust and well-characterized, but its oriented phase exists over a relatively narrow temperature range (32−36 °C). Orientation can be obtained over a wider range of temperatures by using modified lipids or detergents.40−42 Adding cholester-

INTRODUCTION Divalent cations are critical for the functions of many biomolecules and therefore must be included in samples for biophysical characterization and structure determination. Many RNAs require Mg2+ at concentrations of 1−10 mM for proper folding,1,2 binding to a target molecule, or ribozyme activity.3,4 Many proteins also require divalent cations for activity, e.g. zinc fingers in nucleoporins,5,6 and DNA binding proteins,7 the Mg2+-dependent enzymes glucose-6-phosphate dehydrogenase8 and bovine heart glycogen synthase D,9 and the many calciumdependent proteins such as calmodulin.10−12 Other proteins such as the annexins13−15 and the membrane protein phospholamban16−19 can assume different conformations depending in part on the divalent cation concentration, making membrane mimetics that are tolerant of a range of salt concentrations essential to their characterization. Membrane mimetics enable biophysical and structural characterization of membrane proteins in their native states, as has been productively exploited using both solid-state and solution NMR. Many mixtures of lipids and detergents have been used for this purpose, with a variety of structures being formed, including micelles, vesicles, and bilayers. The choice of system depends on the native environment of the protein and its sensitivity to the membrane composition.20 Membrane proteins can be solubilized in micelles or vesicles, but the resulting samples do not always show full biological activity as has been demonstrated for proteases and protein−protein interactions.21−23 Enzymatic metalloproteins, such as the matrix metalloproteinase family, require both divalent cations24 and a bilayer environment to ensure native activity, as small micelles © 2012 American Chemical Society

Received: September 7, 2011 Revised: May 1, 2012 Published: May 1, 2012 7788

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Figure 1. 31P spectra recorded on a 400 MHz Bruker DRX instrument. Samples were doped to 13.4% cholesterol sulfate with respect to moles of DMPC and were 15% w/v lipid with q = 2.6 (A) and q = 3.5 (B). Samples were doped with the divalent cations Ca2+ and Mg2+ 10, 50, and 100 mM and externally referenced to 85% H3PO4. Spectra are colored according to the phase(s) present in the sample as follows: black: fast-tumbling bicelles (isotropic); blue: wormlike micelles or “ribbons” (chiral nematic); green: vesicles; orange: mixed phase (wormlike micelles + vesicles); red: unaligned macroscopically ordered phase. (A) At 25 °C, all the q = 2.6 samples are fully in the isotropic bicelle phase. As the temperature is increased, a magnetically aligned phase is observed, as indicated by the spectrum splitting into two peaks. The high viscosity of this phase is consistent with the chiral nematic phase previously identified by optical microscopy, small-angle neutron scattering, and TEM measurements.39,51 This occurs at the isotropic-to-magnetically aligned transition temperature Tm. At the concentrations studied, Ca2+ has no effect on Tm, and Mg2+ does so only at a concentration of 100 mM. At the higher phase transition temperature Tv, the magnetically aligned phase gives way to structures characterized by more extended lamellae. In this region of the phase diagram, multilamelllar vesicles (MLVs) are formed by undoped DMPC/DHPC mixtures as well as bicelle mixtures containing negatively charged lipids, while unilamellar vesicles (ULVs) have been observed in mixtures doped with either positively charged lanthanide ions of Ca2+.52 In the mixtures described here, both negatively charged lipids and divalent cations are present, and it is not possible to distinguish MLVs from ULVs using NMR methods alone. Therefore, we will refer to this phase as unaligned vesicles pending a more complete characterization. Mixed phases composed of wormlike micelles and vesicles are also observed. Although neither metal ion significantly affects Tv, both influence the composition of the high-temperature phase. (B) For the more strongly aligned q = 3.5 bicelle mixture, the effect of the divalent metal ions is more pronounced. All spectra were compared to deuterium quadrupolar splitting data (Figure 3 and Figure S1) to help determine the phases present. Hash marks indicate where peaks were cut off for space reasons.

ol43,44 widens the temperature range of the aligned phase and increases the degree of alignment but can have problematic interactions with solutes. Addition of charged amphiphiles such as CTAB or SDS to bicelle mixtures stabilizes the samples with respect to addition of solutes and prevents phase separation by helping to counteract the detrimental effect of high salt

concentration,45 while adding cholesterol sulfate (CS) appears to combine both effects.46 Addition of solutes also plays a role; small, uncharged molecules47 and divalent cations48 have been found to alter the bicelle alignment and ordering, while addition of lanthanide ions can change the orientation of the alignment by 90°.34 7789

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Figure 2. 31P spectra recorded on a 400 MHz Bruker DRX instrument. Samples were doped to 13.4% cholesterol sulfate with respect to moles of DMPC and were 15% w/v lipid with q = 2.6 (A) and q = 3.5 (B). Samples were doped with the divalent cations Zn2+ and Cd2+ to 10 and 100 mM and externally referenced to 85% H3PO4. Spectra are colored according to the phase(s) present in the sample as follows: black: fast-tumbling bicelles (isotropic); blue: wormlike micelles or “ribbons” (chiral nematic); green: vesicles; orange: mixed phase (wormlike micelles + vesicles); red: unaligned macroscopically ordered phase; purple: perforated vesicles. (A) As the temperature is increased, a magnetically aligned phase is observed, as indicated by the spectrum splitting into two peaks. This occurs at the isotropic-to-magnetically aligned transition temperature Tm. At the concentrations studied, Zn2+ raises the Tm at 50 mM and Cd2+ does so at 50 and 100 mM. At the higher phase transition temperature Tv, the wormlike micelle-to-vesicle transition occurs, yielding either vesicles or a mixed phase consisting of wormlike micelles and vesicles. Zn2+ lowers the Tv at 50 and 100 mM and changes the composition of the high temperature phase to vesicles at 55 °C. (B) For the more strongly aligned q = 3.5 bicelle mixture, all concentrations of Zn2+ and Ca2+ raise the Tm. Although 100 mM Zn2+ is the only ion concentration that lowers the Tv, all concentrations affect the composition of the high-temperature phase. All spectra were compared to deuterium quadrupolar splitting data (Figure 3 and Figure S1) to help determine the phases present. Hash marks indicate where the spectra were cut in order to fit it in the figure.

mimetics, the effect of these ions on the aligned phases must be investigated in order to perform either solution-state RDC measurements or solid-state NMR experiments on cationdependent biomolecules. Furthermore, divalent cations may be used to modulate the membrane phase morphology and degree of alignment for a wider range of applications. Here we investigate the phase behavior of DMPC/DHPC/CS bicelle mixtures in the presence of biologically relevant divalent cations. The effects of cadmium and the use of EDTA in

Low salt concentrations can be assumed to cause minimal perturbation of the oriented phase; however, some biomolecules require surprisingly high concentrations of divalent cations for optimal invitro activity. For example, the calcium binding protein annexin does not reach full saturation until 40− 50 mM calcium,14 and the M1 RNA which is the catalytic subunit of RNase P has its maximum activity at 100 mM magnesium.49 Because bicelle mixtures are used both as weakly orienting media for soluble molecules and as membrane 7790

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calcium samples were also tested in order to elucidate the mechanism of interaction with the aligned phase. The Mg2+ and Ca2+ ions have been shown to slightly increase the size, alignment, and temperature stability of DMPC and DCPC bicelles,48 but a systematic study of their effect on the temperature-stabilized DMPC/DHPC/CS bicelle mixtures over a range of concentrations needed for biological activity has not previously been made.

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EXPERIMENTAL SECTION

Bicelle Mixture Preparation. The 1,2-dimyristoyl-sn-glycero-3phosphocholine (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine were purchased from Avanti Polar Lipids (Alabaster, AL) in chloroform. Dissolved lipids were combined in the proportions needed for the desired q = [DMPC]/[DHPC] and mass percent, dried under a nitrogen stream for 8 h, and lyophilized for 24 h. Cholesterol sulfate was added to 13.4% with respect to moles of DMPC, and the samples were hydrated in a solution of 10% D2O, 10 mM Tris buffer, pH 6.8, and 0.05% NaN3. Samples were allowed to hydrate for 3−5 days, and the divalent cations were added. The samples were temperature cycled between 37 and 4 °C three times. NMR Spectroscopy. 2H and 31P spectra were recorded on a 400 MHz Bruker DRX spectrometer. After preparation, the samples were allowed to warm to room temperature outside of the magnetic field. Spectra were collected by starting with the lowest temperature and raising it through all sampled temperatures. The samples were allowed to equilibrate for 30 min at each temperature prior to data acquisition. For 2H spectra 16 transients were collected via the lock channel. 31P spectra were collected with a Waltz-16 broadband 1H decoupling sequence,50 and 1024 scans were collected. Approximate bicelle domain size was determined as discussed in Arnold et al.48

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RESULTS AND DISCUSSION P NMR. In order to determine the degree of sample ordering and alignment, both 2H and 31P spectra were recorded for 15% lipid w/v bicelle mixture samples containing 13.4 mol % of CS with respect to DMPC and each of the biologically relevant divalent cations Ca2+, Mg2+, and Zn2+ at varying concentrations using the chloride salts. Spectra of Cd2+-doped samples with Cd2+ were also collected for comparison, providing additional insight into the mechanism for the effects on the transition temperatures and phases. 31P spectra were recorded for samples with q = 2.6 and 3.5. Ions were added to a concentration of 10, 50, and 100 mM for Ca2+, Mg2+, and Zn2+. Spectra were taken in 10 deg increments from 25 to 55 °C. In Figures 1 and 2 the spectra are shown with colors designating the phase. In cases where it was not clear from this data whether the phase was aligned, or if it was a mixed phase, the deuterium spectra shown in Supporting Information Figure 1 were used for clarification. Calcium and Magnesium Ion Effects. Calcium and magnesium ions, both of which act as hard Lewis acids,53 had similar effects on the bicelle mixtures as shown by 31P spectra. Figure 1 (top) shows a comparison among all the low DMPC to DHPC (q = 2.6) ratio samples, with ion concentration (Ca2+ and Mg2+) varying from right to left and temperature increasing from bottom to top. All samples were compared to a reference sample that was not doped with any metal ions. For all of the q = 2.6 samples, single narrow resonance is visible at 25 °C, indicating that the sample is below the isotropic-to-magnetically aligned phase transition (Tm) temperature and is undergoing randomized isotropic motion. When the temperature is increased to 35 and 45 °C, two peaks are observed, the lowfield peak corresponding to DHPC and the high-field peak corresponding to DMPC.54 While the intensities and widths of 31

Figure 3. Comparison of deuterium quadrupole splitting in 10% D2O/ 90% H2O, q = 3.5 DMPC/DHPC bicelle mixtures of total lipid content 15% w/v with 13.4% cholesterol sulfate doping, measured in a 400 MHz Bruker DRX instrument. Representative spectra are shown for each ion, demonstrating the utility of these systems for structural studies of nucleic acids and proteins that require divalent cations. (A) and (B) (1 and 100 mM, respectively) show representative spectra for magnesium chloride doped lipids. (C) and (D) (1 and 50 mM, respectively) show representative spectra for calcium chloride doped lipids. (E) shows representative spectra for zinc chloride at 10 mM, and (F) shows representative spectra for cadmium chloride doped lipids. Spectra are colored according to the phase(s) present in the sample as follows: black: fast-tumbling bicelles; blue: wormlike micelles; green: vesicles; orange: wormlike micelles + vesicles. The Tm can be seen when the single isotropic peak splits into two peaks representing alignment of the wormlike micelle phase. Tv can be observed when the two split peaks re-form a single peak isotropic peak representing a vesicle phase or a combination showing three peaks representing a mixed phase of wormlike micelles and vesicles. A full set of deuterium spectra for all concentrations of ions can be found in Figure S1. 7791

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Table 1. Effects of Calcium on “Bicelle” Domain Size

q Φ

no M2+

10 mM CaCl2

50 mM CaCl2

100 mM CaCl2

50 mM CaCl2 + EDTA

3.11 430

3.65 500

3.74 510

4.01 540

2.87 400

However, the phase that is present in the Ca2+ and Mg2+ ion doped samples is not identical to that of the undoped sample. A broader high-field peak indicates a higher degree of mosaicity.48 The 2H NMR (Figure S1) confirms that this is a magnetically aligned phase and not simply a macroscopically ordered nonaligned phase. The effect on Tv for the q = 3.5 samples is similar to those observed in the q = 2.6 samples. Here the calcium lowers the Tv to 45 °C. However, when the temperature is increased to 55 °C, the same fast-tumbling bicelle and wormlike micelle phase observed in the q = 2.6 sample is present. Mg2+ and Zn2+ do not affect the Tv. However, Mg2+ does affect the phases and as with the Ca2+ goes into a mixed phase. 10 mM Zn2+ did not affect the phase at 55 °C sample. Zinc and Cadmium Ion Effects. The effects on the bicelle mixtures from the divalent ions of zinc, a borderline hard Lewis acid,53 and cadmium, a soft Lewis acid,53 were similar to each other but varied significantly from those of calcium and magnesium ions. In Figure 2, for all ion concentrations of the low DMPC to DHPC (q = 2.6) ratio samples, a single narrow resonance is visible at 25 °C, showing that the sample is below the bicelle-to-magnetically aligned phase transition (Tm) temperature and is undergoing randomized isotropic motion. When the temperature is increased to 35 and 45 °C, two peaks are observed: the low-field peak corresponding to DHPC and high-field peak corresponding to DMPC. 54 While the intensities and widths of the DMPC signals vary with ion concentration and temperature, all follow the same trend. The broad powder patterns in the 100 mM Mg2+ sample are representative of an unaligned, macroscopically ordered phase. At 55 °C the samples with no ion and 10 mM Mg2+ reach Tv, and a third peak becomes apparent. In this phase wormlike micelles and vesicles coexist.54 For Zn2+, the single isotropic peak and broad powder pattern at 55 °C is indicative of fasttumbling bicelles and a weakly aligned “bicelle” or chiral nematic phase, respectively.55,56 Tv is affected by these divalent ions to a much larger extent. In the q = 2.6 sample, adding 100 mM Zn2+ lowers Tv to 45 °C. 10 mM Zn2+ does not affect the Tv significantly, but it does alter the composition of the high-temperature phase. A distinct isotropic peak due to fast-tumbling bicelles and a high-field broad powder pattern peak, due to aligned bilayers are visible, as opposed to the three distinct peaks in the sample containing no divalent metal ions. For the high DMPC to DHPC ratio sample (q = 3.5), a macroscopically ordered phase is present at room temperature for 10 mM Zn2+ and Cd2+. The peak intensity and width of the high-field peak varies significantly. All samples at 35 °C were in the chiral nematic phase. When the temperature was raised to

Table 2. Effects of Divalent Ions on ”Bicelle” Domain Size

q Φ

no M2+

50 mM CaCl2

50 mM MgCl2

50 mM ZnCl2

50 mM CdCl2

3.11 430

3.74 510

3.26 450

2.73 390

3.00 420

the DMPC signals vary with ion concentration and temperature, all follow the same trend. The broad powder patterns in the 100 mM Mg2+ sample are representative of an unaligned, macroscopically ordered phase. At 55 °C the samples with no divalent ions and 10 mM Mg2+ reach Tv, and a third peak becomes apparent. In this phase wormlike micelles and vesicles coexist.54 For Ca2+, the single isotropic peak and broad powder pattern at 55 °C are indicative of fast-tumbling bicelles and a weakly aligned chiral nematic phase, respectively.55,56 Tm in the q = 2.6 sample is only affected by magnesium ions and only when the concentration reaches 100 mM. At this concentration, although a macroscopically ordered aligned phase is observed, the differences in the spectrum show that the composition of the phase is altered. This result is relevant to structural studies of nucleic acids and nucleic acid-binding proteins, as 10 mM is more than sufficient to ensure physiologically relevant conditions, and the Tm is unaffected at this concentration. The composition of the high temperature phase is affected by the divalent ions to a much larger extent. 10 mM Ca2+ does not affect the Tv significantly, but it does alter the composition of the higher temperature phases. A distinct isotropic peak due to fast-tumbling bicelles and a high-field broad powder pattern peak, due to aligned bilayers are visible, as opposed to the three distinct peaks in the sample containing no divalent ions. The 10 mM Mg2+ sample has both the two distinct isotropic peaks and the broad powder pattern, indicating a mixed phase. For the high DMPC to DHPC ratio sample (q = 3.5), a macroscopically ordered phase is present at room temperature for all magnesium and calcium ion concentrations. However, at 100 mM magnesium, the phase is still unaligned. All q = 3.5 samples at 35 °C were in the chiral nematic phase. When the temperature was raised to 55 °C, all the calcium and magnesium samples reached Tv and transitioned into either a mixed wormlike micelle and vesicle phase or a vesicle-only phase. The q = 3.5 Ca2+ samples are all aligned at 25 °C. Table 3. Deuterium Quadrupolar Coupling (Hz)

a

temp (°C)

no M2+

1 mM CaCl2

10 mM CaCl2

50 mM CaCl2

50 mM CaCl2, EDTA

100 mM CaCl2

1 mM MgCl2

10 mM MgCl2

50 mM MgCl2

100 mM MgCl2

25 30 35 40 45 50 55

1.6 10.4 19.1 22.2 24.0 a 0

5.1 19.5 24.2 26.6 30.8 39.4 a

2.5 12.3 48.0 57.4 30.1 a b

11.2 12.6 41.3 57.4 7.4 a a

0 10.9 19.6 25.0 31.03 a a

0 6.2 18.1 23.4 27.5 a a

4.9 9.0 15.3 17.3 23.4 24.0 29.5

2.6 35.2 41.8 48.6 56.5 a a

2.0 9.0 47.3 57.5 59.7 a a

14.0 21.5 28.9 33.3 a b b

Neither magnetically aligned nor isotropic phase. bData not taken. 7792

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Table 4. Deuterium Quadrupolar Coupling (Hz)

a

temp (°C)

no ion

1 mM ZnCl2

10 mM ZnCl2

50 mM ZnCl2

100 mM ZnCl2

1 mM CdCl2

10 mM CdCl2

50 mM CdCl2

25 30 35 40 45 50 55

1.6 10.4 19.1 22.2 24.0 a 0

1.6 9.9 20.2 23.0 25.9 a a

0 13.7 20.2 28.6 27.7 a a

0 2.0 14.6 19.1 a a b

0 9.0 19.3 a a a a

3.2 8.0 24.4 27.5 31.5 36.4 a

0 12.3 23.4 28.7 33.5 a a

0 14.1 21.9 27.8 41.3 a a

Neither magnetically aligned nor isotropic phase. bData not taken.

45 °C, the 100 mM Zn2+ sample reached Tv and transitioned into a mixed wormlike micelle/vesicle phase. The 10 mM Zn2+ and both Cd2+ samples remained in a wormlike micelle phase. At 55 °C, all zinc and cadmium doped samples transitioned into a mixed phase. In several of the Zn2+ samples an extremely narrow isotropic peak is visible, corresponding to a small molecule-sized phosphorus compound, possibly indicating interaction of the Zn2+ ion with a small number of lipid molecules. The Zn2+ ions do raise the Tm. At 10 mM the sample is macroscopically ordered but mostly unaligned, as indicated by the broad powder pattern in the high-field peak. Although similar to the other spectra, the peak width increases and the shoulder becomes more prominent. The 2H spectra shown in Figure 3 support this conclusion. The effect on Tv for the q = 3.5 is similar to that in the 2.6; Zn2+ does not affect the Tv at 10 mM. However, the 100 mM sample had a lowered Tv, and it formed a different phase from the rest, reaching Th where the holes in the aligned vesicles disappear due to miscibility of DHPC in DMPC. For a simple DMPC/DHPC system this is only seen when q is above 5.41 “Bicelle” Size. A semiquantitative analysis of the 31P spectra can be used to determine an estimate of the size of the domains in the aligned phase. The areas of the DMPC and DHPC peaks give a q factor. We used the method described in Arnold et al.48 to determine the aligned phase domain size for all ions at 50 mM and calcium at each concentration measured. Each q was determined through three integrations at the temperatures where the aligned phase was present as determined by Figures 1 and 2 and Figure S2. The average q was used to calculate the radius of the domain which is listed in Tables 1 and 2. As the concentration of Ca2+ increases, the size does as well. The approximate domain radius was calculated for all cations at 50 mM since the largest differences in interactions were present at this concentration. As expected, Ca2+ significantly increases the size of the domains. Cd2+ slightly decreases the domain size and Zn2+ decreases the size further. When EDTA is added to the calcium samples the domain size decreased, confirming that the calcium ions interact with the lipids to affect the alignment. 2 H NMR. 2H spectra were recorded for samples of q = 3.5 with cations at the concentrations as shown in Figure 3. Spectra were recorded in 5 °C increments from 25 to 55 °C. Samples were allowed to equilibrate for 30 min prior to acquisition, and a second spectrum was acquired 5 min after the first to check for full equilibration. Quadrupolar splitting is seen from 25 to 45 °C in the DMPC/DHPC/CS bicelle mixtures. The increase in the observed coupling value with increasing temperature is consistent with increasing domain size. Calcium and Magnesium Ion Effects. Adding 1 mM Ca2+ or Mg2+ increases the upper limit of this range to 50 and 55 °C, respectively. Further addition of Ca2+ or Mg2+ gives the same range of alignment as the undoped sample. Tables 3 and 4 list

the quadrupolar splittings for each sample. Representative spectra of samples containing each ion are shown in Figure 3, and the complete set can be found in the Supporting Information (Figure S1). As shown in Figure 4, the magnitudes of the splittings, reflecting the degree of orientation, are also affected by the type and concentration of divalent ions. Calcium ions increase the magnitudes of the splittings for all temperatures and concentrations. Magnesium at 1 mM had very similar splittings to the undoped samples, indicating that structures of RNAs requiring this concentration can be undertaken with minimal perturbation of the orienting media. At 25 °C, 50 and 100 mM Mg2+ did not affect the alignment. When the temperature was raised to 30 °C, the 10 mM sample has significantly larger couplings, and when the temperature was raised to 35 °C, this was seen in the 50 mM sample as well. The 2H spectra are in good agreement with the 31P spectra. Ca2+ and Mg2+ increase the degree of alignment as shown by the increased quadrupolar splittings. The transition into a mixed phase occurs at 45 °C for the 10 and 100 mM Ca2+ samples according to the 31P spectra. The 2H spectra show that for 10 and 50 mM Ca2+ samples, although the mixed phase is present, there is still a large degree of alignment up until 45 °C with a single broad peak being present at 50 °C (Figure 3 and Figure S1) indicating a nonaligned phase. In the 10 mM Mg2+ sample, the 31P spectra showed that the transition from chiral nematic to a mixed phase occurred between 45 and 55 °C. The 2 H show that at 50 °C the peaks separate into two separate aligned phases which become even more prominent at 55 °C. At higher concentrations of Mg2+, the 31P spectra looked more similar to the undoped chiral nematic spectra; this was also true of the 2H spectra. A gradual increase in quadrupolar couplings occurred until at 55 °C a single broad isotropic peak was present . Zinc and Cadmium Ion Effects. The addition of Zn2+ raises the Tm to 30 °C for 50 mM and 35 °C for the addition of 100 mM. Zn2+ also lowers the transition to the mixed phase to 40 and 35 °C. This gave an extremely narrow range of alignment for the 50 and 100 mM Zn2+ samples, indicating that NMR experiments are best performed at lower Zn 2+ concentration if possible. The addition of 1 mM Cd2+ had no effect on Tm and raised the Tv. At higher concentrations of 10 and 50 mM cadmium ions, the Tm was raised and the Tv was lowered, decreasing the range at which the magnetically aligned phase is present. Tables 3 and 4 list the quadrupolar splittings for each sample. Representative spectra of samples containing each ion are shown in Figure 3, and the complete set can be found in the Supporting Information (Figure S1). As shown in Figure 4, the magnitudes of the splittings, reflecting the degree of orientation, are also affected by the type and concentration of divalent ions. The 10 mM Zn2+ sample slightly increased the 7793

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Figure 5. Comparison of the bulk deuterium quadrupole splitting in 10% D2O/90% H2O, q = 3.5 DMPC/DHPC bicelles mixtures of total lipid content 15% w/v with 13.4% cholesterol sulfate doping and the divalent cations. (A) 10 mM doped samples (B) 50 mM doped samples, collected in a 400 MHz Bruker DRX instrument. See Figure S3 for 1 and 100 mM samples.

magnetically aligned phase is present by raising the Tm and lowering Tv. As discussed in the 31P results section, 10 mM Zn2+ or Cd2+ is mostly unaligned but macroscopically ordered, giving rise to three extremely broad peaks as shown in Figure 3 and Figure S1. Discussion of Trends in 2H Spectra. The biggest difference in splittings was observed in the 10 and 50 mM doped samples. Figure 5 clearly shows that while in the magnetically aligned phase Ca2+ and Mg2+ greatly increase the alignment, while Zn2+ and Cd2+ do not show a large effect (1 and 100 mM, see Figure S1). The semiquantititative determination of “bicelle” domain size (Table 2) suggests that the increase in alignment is caused by an increase in domain size. Zn2+ and Cd2+ both decrease the size of the domains slightly while Ca2+ and Mg2+ increase the size of the domains. The increase in size can be linked with the strength of the interactions between the metal ion and the phosphate group of the lipids. Magnesium and calcium divalent ions are harder acids, and both sulfate and phosphate are hard bases.53 According to HSAB theory, hard acids preferentially associate with hard bases,53 so the calcium and magnesium ions will have a greater affinity for the aligned phase than softer zinc and cadmium ions. The lower hardness values of Zn2+ and Cd2+ and their subsequent lower affinity for the phosphate and sulfate groups on the membranes cause nonspecific electrostatic interactions that destabilize the magnetically aligned phase, increasing Tm and decreasing Tv.

Figure 4. Comparison of the bulk deuterium quadrupole splitting in 10% D2O/90% H2O, q = 3.5 DMPC/DHPC bicelle mixtures of total lipid content 15% w/v with 13.4% cholesterol sulfate doping and the divalent cations. Ca2+ (A), Mg2+ (B), Zng2+ (C), and Cdg2+ (D), collected in a 400 MHz Bruker DRX instrument.

magnitude, 50 mM reduced the magnitude slightly, and 10 mM had no significant effect on the magnitude of the couplings. The 2H spectra are in good agreement with the 31P spectra. The Zn2+ and Cd2+ have little effect on the degree of alignment but produce a much smaller temperature range where the 7794

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CONCLUSION We have shown that divalent cations can be used in samples with DMPC/DHPC/CS bicelle mixtures. Many biological macromolecules are both temperature-sensitive and require divalent cations to be present in buffers, necessitating an alignment medium that can be used at room temperature and is tolerant of the cations at a variety of concentrations. Many zincdependent proteins require only ∼10 μM in solution, while calcium and magnesium ions are needed for a variety of protein and nucleic acid structure determinations over a larger concentration range. At low ion concentrations, minimal perturbation of the magnetically aligned phase is observed, while higher concentrations affect both the phases formed and the transition temperatures. This is consistent with the finding that divalent cations can change the composition of lamellar phases by influencing the interaction between membrane surfaces.57 In DMPC/DHPC bicelle mixtures, Ca2+ increases the alignment over all temperatures and extends the temperature range that the aligned phase is present. While small concentrations of Mg2+ decreased the degree of alignment slightly, it did not affect the transition temperatures dramatically. At higher concentrations it increased the alignment. All three biologically relevant cations can be used in conjunction with the DMPC/DHPC/CS bicelle mixtures at temperatures down to 30 °C, allowing structural determination experiments to be carried out on temperature-sensitive biomolecules. As expected, when EDTA was added to the calcium doped samples, it reduced the alignment and size of the domains in the aligned phase to predoped conditions, confirming the presence of a direct interaction between the metal ion and the lipid phase. In a previous study, using chelated lipids with copper doped samples was shown to reduce the amount of free copper, while maintaining the decrease in relaxation time that is characteristic of the paramagnetic ions.58,59 Although the utility of the particular sample preparation described here is limited to the many situations where the divalent ions interact with the molecule of interest in a biologically relevant way or has no interaction, it should be noted that future more general implementations could be performed by using metal cations specifically chelated to lipid molecules. In summary, we have shown that divalent cations may be used to manipulate the phase behavior of bicelle mixtures.

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REFERENCES

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ASSOCIATED CONTENT

* Supporting Information S

Additional 2H and 31P spectra for the different bicelle mixtures tested. This material is available free of charge via the Internet at http://pubs.acs.org.

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Article

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Andrej Lupták and A. S. Borovik for helpful discussions and Phil Dennison for excellent NMR facility management. This work was supported by NSF CAREER CHE-0847375 and the UC Cancer Research Coordinating Committee. 7795

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