1H NMR MAS Investigations of Phase Behavior in Lipid Membranes

Chapter 16

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1H

NMR MAS Investigations of Phase Behavior in Lipid Membranes Holly C. Gaede* Department of Chemistry, Texas A&M University, College Station, Texas 77843 *E-mail: [email protected]

Solid-state nuclear magnetic resonance experiments that characterize the phase behavior of phospholipids are described. In particular 1H magic angle spinning NMR spectroscopy experiments on mixtures of 1,2-dimyristoyl-sn-glycero-3phosphocholine (DMPC) and cholesterol can be used to determine the phases present at different temperatures and compositions.

Introduction Phospholipids are the most abundant component of biomembranes. As amphipathic molecules with both polar headgroups and non-polar hydrocarbon tails, these phospholipids spontaneously form bilayer sheets 3 - 4 nm thick, with the polar headgroups facing outward toward the water and the hydrocarbon chains forming a hydrophobic core. Another important membrane lipid in mammalian cell membranes is cholesterol. Insertion of the rigid cholesterol molecule into a bilayer can decrease the mobility of the first few hydrocarbon groups along the phospholipid chain, decreasing membrane fluidity. Conversely, cholesterol can interfere with the close packing of these acyl chains, inhibiting its transition to a crystalline state. Other components of cell membranes include integral and peripheral membrane proteins and carbohydrates. All of the components of the membrane together make up what was termed the “fluid mosaic model.” In the late 1980s, the presence of submicron-size domains in cell membranes that are enriched in cholesterol was proposed in areas now called “lipid rafts.” It is now clear that lipids are laterally segregated throughout the cell (1). Because certain proteins © 2013 American Chemical Society In NMR Spectroscopy in the Undergraduate Curriculum; Soulsby, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


involved in cellular signal processes have been shown to associate with lipid rafts, it is thought that these rafts may play a role in cell signal transduction (2). Rafts have also been suggested to play a role in the entrance of certain pathogens, including HIV (3), into the cell. Being able to understand and regulate lipid rafts may open methods for preventing or alleviating a variety of infectious diseases. However, the existence of lipid rafts remains controversial, in part because lipid rafts are difficult to observe in actual membranes (4).

Lipid Phases To understand the phenomenon of lipid rafts, simpler model systems without proteins are often studied so as to develop phase diagrams. In biological membranes, lipids are in the liquid crystalline state, where the molecules are free to rotate about their long axes and laterally diffuse in the plane of the membrane. This state has also been referred to as the liquid disordered state (ld), which is characterized by rapid gauche/trans isomerizations of the lipid hydrocarbon chain. If the temperature is lowered below the phase transition temperature, Tm, the lipid enters the gel state, also known as the solid ordered state (so). The hydrocarbon chain is more rigid and ordered in this state, with strong van der Waals interactions among the chains, and the gauche/trans isomerization is mostly suppressed. The rotational diffusion about the bilayer normal is slow, and the lateral diffusion in the membrane plane is slowed by orders of magnitude compared to the ld state. In systems containing saturated lipids and cholesterol, a third phase has been observed, called the liquid-ordered state (lo). The exact nature of this state is still under debate, but early evidence shows that cholesterol hinders gauche rotamer formation at certain carbons close to the carbonyl because of the cholesterol’s preferred location near the headgroup region. However, rates of rotational and lateral diffusion remain largely unchanged from the ld state (5). Biological rafts are thought to be small accumulations of liquid ordered regions surrounded by pools of liquid disordered region, though these regions are dynamic and the composition may change. The size and lifetime of the rafts in real biological membranes is still unknown. Several different physical techniques have been used to study the phase behavior of lipids. Nuclear magnetic resonance (NMR) spectroscopy is an attractive method to use since these molecules contain many nuclei that are accessible to NMR spectroscopic investigation, including hydrogen, carbon and phosphorous, and therefore no phase-perturbing labels are required. Though conventional solution-state NMR spectra may be readily obtained for a lipid dissolved in a solvent like chloroform or methanol, these spectra do not give information about the lipids in a membrane environment. Because these bilayer samples do not tumble isotropically like small molecules in solution, conventional solution-state NMR spectra will show broad featureless lines because of the anisotropic chemical shift and dipole-dipole interactions. However, solid-state NMR techniques, such as magic angle spinning, can be used to modulate these interactions, discussed in detail below, to gain structural and dynamic information. 246 In NMR Spectroscopy in the Undergraduate Curriculum; Soulsby, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


This chapter will outline a series of NMR experiments used in a physical chemistry laboratory, but appropriate for other upper division laboratories, that investigates the phase behavior of the lipids 1,2-dimyristoyl-sn-glycero3-phosphocholine (DMPC) and cholesterol using magic angle spinning NMR spectroscopy. These experiments not only introduce students to the important biophysical system of lipids, but also expose them to the increasingly important technique of magic angle spinning NMR.

Anisotropic Magnetic Interactions This section will introduce the important anisotropic –angularly dependent – interactions that, while averaged in the rapid isotropic tumbling of molecules in solution, generally cause additional broadening in solid samples.

Dipole−Dipole Interaction The dipolar interaction results from interaction of one nuclear spin (I1) with the magnetic field generated by another nuclear spin (I2), and vice versa. In general, the energy of interaction between two magnetic dipoles separated by a distance r is given by

and

where μ0 is the permeability constant. The Hamiltonian is obtained by replacing the vectors and by their corresponding operators and , where γ1 and γ2 are the gyromagnetic ratios of nuclei 1 and 2. Specifically, the interaction between dipolar coupled spins is governed by the dipolar Hamiltonian.

Equation 2 can be expanded into several terms, each containing a spin part and a spatial part that can be considered separately. For homonuclear spins (e.g., both protons) in high field, it simplifies to

247 In NMR Spectroscopy in the Undergraduate Curriculum; Soulsby, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


The group of constants is called dipolar coupling constant and is defined as

In solution molecules reorient quickly and the nuclear spins feel a time average of the spatial part of the dipolar interaction over all orientations, which is zero. Therefore, the dipolar effects only manifest themselves through relaxation. These relaxation effects are exploited in important two-dimensional NMR spectroscopic techniques that include NOESY. However, the dipolar interaction can cause severe broadening in solids, where every spin is coupled to every other spin. The degree of broadening depends on the nuclei involved and the distance between them (Table 1).

Table 1. Dipolar Coupling Constants, RDD Nuclei

Distance (Å)

Coupling (kHz)

1H

- 1H

1

120

1H

-

13C

1

30

1H

-

13C

2

3.8

The coupling between two protons is typically 120 kHz, equivalent to 240 ppm in a spectrometer operating with a proton frequency of 500 MHz! This broadening obscures the much smaller chemical shift and J-coupling interactions which are exploited in solution for structural determination. However, this dipolar coupling can also be a rich source of information. Chemical Shift Anisotropy Another important source of broadening in solids arises from chemical shift anisotropy. Recall from solution NMR that the nucleus is shielded from the external magnetic field by the electrons. This phenomenon is why an aldehyde proton has a different characteristic frequency than a methyl proton. The chemical shift is critically important in structural elucidation. In solids, both the local electronic environment and the orientation of the molecule relative to the magnetic field play a role in the amount of shielding experienced by a nucleus. The orientation is important because the electronic distribution around a nucleus within a molecule is rarely spherically symmetrical. In general, a nuclear environment will have its shielding characterized by three distinct values. These three values are referred to as the principal components and occur for orientations specified by the principal axes. The three components are summarized by the shielding tensor, σ. 248 In NMR Spectroscopy in the Undergraduate Curriculum; Soulsby, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


The principal components are assigned such that σ11≤σ22≤σ33, and the observed shielding σobs is denoted

where the angles, θj, are those between the principal components and the magnetic field. In solution, the molecule typically tumbles rapidly and only the average chemical shift is observed. The isotropic shielding, σiso, which is observed in solution NMR, is given by

Equation 6 and equation 7 can be combined to give

As a result a randomly oriented solid would result in a powder spectrum with signal intensities corresponding to the probability of a particular orientation. However, neglecting the dipolar interaction discussed above, it is possible to observe narrow lines if all the nuclei have the same orientation relative to the magnetic field, as in the case of single crystals or otherwise oriented samples. Only two extrema in the powder patterns will be observed for a molecule with axial symmetry, as is case for 31P NMR of lipid headgroup resonances. A molecule lacking this symmetry will have three extrema. Quadrupolar Interactions A third interaction can cause broadening for certain nuclei in solids. The most commonly investigated nuclei in lipids (1H, 13C, and 31P) are all spin 1/2 nuclei, and have a spherical charge distribution. As a result, spin 1/2 nuclei do not interact with electric field gradients (spatial variations in the electric field) found in the sample. However, quadrupolar nuclei have a spin > 1/2, and a non-spherical electric charge distribution. This anisotropy can cause extra splitting and broadening, but is not a factor in the following experiments. 249 In NMR Spectroscopy in the Undergraduate Curriculum; Soulsby, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.

Magic-Angle Spinning


The anisotropic interactions described above have a (3cos2θ-1) term called the second-order Legendre Polynomial, P2(θ) that is averaged by rapid isotropic tumbling in solution. The same effect can be achieved in the solid state if the sample is rotated rapidly at an angle relative to the magnetic field such that . This angle, θ=54.7°, is called the magic angle and as a result the technique is called magic angle spinning (MAS). In order for MAS to be effective, the spinning frequency must be greater than the magnitude of the anisotropic interactions (i.e., if the broadening due to dipolar coupling is 15 kHz, then the spinning frequency must be greater than 15 kHz). If the anisotropic interactions are greater than the spinning frequency, the spectrum will be reduced to the isotropic resonances (called centerbands) and spinning sidebands, spaced at intervals of the spinning frequencies. These spinning sidebands map out the powder pattern that would be observed in the absence of spinning. These spinning sidebands can be reduced by spinning at a frequency greater than the strength of the anisotropic interactions. MAS and other solid state NMR techniques have been reviewed by Laws (6). For lipid samples in the ld phase at spinning frequencies of 10 kHz, 99% of the proton signal intensities lie in the centerbands. The centerband integral resonance intensities can therefore be related to the number of contributing protons. At 10 kHz, the methylene resonance at 1.3 ppm has a linewidth of ~50 Hz in the ld phase. This linewidth increases dramatically for the lo and so phases, as does the intensity of the sideband resonances. Therefore, the intensities of the lipid resonances, as well as the ratio of sideband to centerband intensity, can be used to determine the phase of the lipid system. MAS is useful for determining the presence of lo and so, but it can be difficult to distinguish between those two phases (5). One advantage of MAS versus other spectral techniques is that MAS requires no special sample preparation or isotopic labeling. Note that the achievable spinning frequencies are still well below the magnitude of typical 1H-1H dipolar interactions, and so 1H detection in a MAS experiment is rare. However, dipolar interactions in liquid crystalline membrane samples are partially averaged by molecular motion, so 1H MAS of these samples allows for high resolution spectra.

Equipment Special NMR sample containers and probes are required for MAS experiments. These probes are no longer limited to specialty research laboratories, and because of the increasing application of MAS to biological samples they are often referred to as High Resolution Magic Angle Spinning (HR MAS) probes. The sample is packed into a special sample container called a rotor, typically a ceramic cylinder with a ceramic or plastic cap with fluted edges. The rotor is spun by passing air or N2 over the cap. Since the membrane samples are liquid 250 In NMR Spectroscopy in the Undergraduate Curriculum; Soulsby, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


crystalline, the samples need to be loaded with a special insert inside the MAS rotor to prevent sample leakage. The sample volume in the inserts depends on the rotor diameter, which in turn is dictated by the NMR probe. For example, Bruker (Billerica, MA) sells inserts in 12 μL or 50 μL volumes for 4 mm diameter rotors. The rotor sits inside a housing called a stator that is inside the probe and whose angle relative to the magnetic field can be precisely adjusted. The stator contains the NMR coil and delivers the spinning gas. Typically, there are two separate gas lines to achieve rapid spinning of the rotor, called bearing and drive. The pressure in each of these lines can be controlled with the controller unit in the console. Modern MAS probes monitor the spinning frequency by optical detection of a mark on the rotor. Spinning frequencies of 1-35 kHz can be achieved routinely, depending on the diameter of the rotor.

Experimental Preparation of DMPC: Cholesterol Samples 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and cholesterol (Avanti Polar Lipids; Alabaster, AL) were dispersed from stock solution of chloroform or methanol into a microcentrifuge tube to give a total lipid mass of 5.0 mg. Note that not all microcentrifuge tubes are compatible with chloroform. The solvent was removed with a gentle stream of nitrogen gas and placed under a vacuum for 30 minutes to remove residual solvent. Deuterated water (Aldrich) (5 μL) was added to the dried lipid to make the sample 50 wt% water. The sample was homogenized by using a vortex mixer and quantitatively transferred to the rotor (4 mm Bruker, B3799) using a centrifuge. Samples were prepared in various DMPC:cholesterol mole ratios, ranging from 1:0 to 1:1. 1H

MAS NMR Experiments

Instrument NMR measurements were performed on a Bruker DMX400 widebore spectrometer (1H operating frequency of 400.1 MHz) running TOPSPIN1.2. Sample spinning at 2-10 kHz was accomplished with a Bruker double-gas-bearing MAS probe for 4 mm rotors. The temperature was controlled with the built-in temperature control unit with house nitrogen gas flowing to the stator through a heat exchanger containing liquid nitrogen.

Spinning-Speed Dependence 1H

NMR spectra were acquired at 25°C at spinning frequencies of 2–10 kHz on a 1:1 DMPC:cholesterol sample. 16k data points were acquired in a one-pulse experiment, with a 90° pulse time of 2.5 μs and a spectral width of 60 ppm. 8 scans were acquired with a 3 s delay between scans. 251 In NMR Spectroscopy in the Undergraduate Curriculum; Soulsby, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.

Temperature Dependence 1H


NMR spectra were acquired at several temperatures from 15–32°C at spinning frequencies of 5 kHz on a number of DMPC:cholesterol samples, with mole ratios ranging from 1:0 to 1:1. 16k data points were acquired in a one-pulse experiment, with a 90° pulse time of 2.5 μs and a spectral width of 11 ppm. 8 scans were acquired with a 3 s delay between scans. The spectra were acquired in automation, with the temperature being systematically incremented with a 900 s equilibration time before signal acquisition.

Results and Discussion DMPC Spectral Assignment The 1H NMR MAS centerband spectrum of 1,2-dimyristoyl-sn-glycero-3phosphocholine (DMPC) along with the chemical shift assignment is given in Figure 1. Though not as high resolution as a corresponding solution spectrum, different functional groups are resolved clearly. In this case, the chemical shift assignment was made on the basis of comparison to literature assignments of other liquid crystalline lipid spectra.

Figure 1. Chemical shift assignment for the 400.1 MHz 1H MAS NMR spectrum of multilamellar liposomes of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) in deuterated water at a rotor spinning frequency of 5 kHz. The structure of DMPC is shown. 252 In NMR Spectroscopy in the Undergraduate Curriculum; Soulsby, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


Though the length and degree of unsaturation of the hydrocarbon chains can vary in phospholipids, the chemical shifts do not vary much from lipid to lipid. Myristoyl is a saturated 14-carbon chain (14:0), which is convenient to study because of amenable physical properties, including relative stability because of the saturated acyl chains and a near-room temperature phase transition. The solution spectrum has very similar chemical shifts, or if desired, students can perform a series of 2D NMR spectroscopy experiments in the solid-state to fully assign the spectrum instead of relying on a comparison to literature assignments (7). It is interesting to note that the methylene in the glycerol group closest to the choline has inequivalent hydrogen atoms. This unusual feature can be difficult to detect since one of the hydrogen atoms (j) is obscured by the resonance signal of HOD. Effect of Rotor Spinning Frequency A stacked plot of 1H MAS NMR spectra of a 1:1 DMPC:cholesterol mixture taken at rotor spinning frequencies of 2.5, 5, and 10 kHz at 25°C is shown in Figure 2. The centerband spectrum is surrounded by spinning sidebands at intervals of the rotor spinning frequency. The intensity ratio of the centerband:spinning sidebands increases with an increased spinning speed, and is shown in the inset to Figure 2. This spinning speed dependence can be used to determine the magnitude of the interactions of the anisotropic interactions. Clearly, a rotor spinning frequency of 2.5 kHz is not sufficient to remove the anisotropic interactions of this sample. Note that a cholesterol-containing sample was chosen for this study because the lipid alone has weaker anisotropic interactions, and the spinning sidebands are harder to observe. Determination of Phase Transition Temperature The spectrum acquired at 10 kHz is the most desirable as far as having the most spectral intensity in the centerband. However, at these spinning speeds, considerable frictional heating can make the temperature of the cooling gas quite different from the actual temperature of the sample. Accurate measurements of temperature require careful temperature calibration (7). In this system, preliminary observation of phase transition behavior of lipids at MAS frequencies of 5 kHz indicated that the thermocouple readings at 5 kHz were accurate, but the actual and recorded temperature varied at MAS spinning frequencies of 10 kHz. Therefore, the study of phase transition was carried out at spinning frequencies of 5 kHz. Figure 3 shows the temperature dependence of the 1H NMR MAS spectra of samples of (A) DMPC and (B) DMPC with 50% cholesterol. The sample of DMPC alone shows a dramatic decrease in signal intensity between 296 and 294 K. In samples below 296 K, the only remaining resolved peaks are from HOD and the headgroup methyl groups, which still retain mobility in the solid-ordered phase. In the sample containing cholesterol, the changes are more subtle. The spectral lines are broader from the overlap of cholesterol resonances and the increased anisotropic interactions. The signal intensity, particularly in the acyl chain region, is also reduced in going from high temperature to low temperature, but never disappears. 253 In NMR Spectroscopy in the Undergraduate Curriculum; Soulsby, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


Figure 2. 1H NMR MAS spectra of a 1:1 DMPC:cholesterol mixture taken at 25 °C at rotor spinning frequencies of a) 10, b) 5, and c) 2.5 kHz. The spectra are plotted in Hertz instead of ppm to emphasize the spinning frequency dependence of the spinning sidebands. Inset: Total sideband and centerband intensity as function of rotor spinning speed.

The signal intensity of the (CH2)n can be plotted to determine the phase transition temperature, Tm. Figure 4 shows the normalized (CH2)n intensity for both the DMPC and the DMPC:cholesterol sample. Again, it is clear that the phase transition between a liquid disordered (ld) phase and a solid ordered (so) phase for DMPC occurs at 295±1 K. 254 In NMR Spectroscopy in the Undergraduate Curriculum; Soulsby, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


Figure 3. Temperature dependence of 1H NMR MAS spectra of A) DMPC at a) 300 K, b) 298 K, c) 296 K, d) 294 K, e) 292 K, f) 290 K, and B) DMPC:cholesterol at a) 305 K, b) 300 K, c) 297 K, d) 294 K, e) 291 K, f) 288 K.

Figure 4. Normalized (CH2)n intensity for DMPC and DMPC:cholesterol samples as function of temperature. 255 In NMR Spectroscopy in the Undergraduate Curriculum; Soulsby, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


This value compares favorably to the literature value of 297 K (8). Conceivably, a first-derivative plot could be used to determine the Tm value more precisely; however, there are too few points acquired to benefit from this approach in this example except perhaps as a pedagogical exercise. Acquisition of more temperature points would allow a more precise determination of Tm. Note that temperature equilibration times of longer than 900 s would be desirable to ensure that the sample temperature readings are accurate, but limited laboratory time forced upon us this compromise. Since there are automation programs to systematically increment the temperature, it would be possible to set up the experiments to run overnight, allowing for more data points with a longer equilibration time for each point. The intensity changes for the cholesterol-containing sample are noticeably different and suggest that there is no phase transition occurring over the temperature range studied. This observation is consistent with the published phase diagram for DMPC:cholesterol (9), which shows that at this ratio of cholesterol, the sample is in the liquid ordered phase (lo) at all temperatures.

Further Studies This experiment can be expanded in several different ways. As mentioned previously, these solid state experiments can be coupled with solution state experiments aimed at spectral assignment of the phospholipid. Alternatively, additional lipids can be investigated with MAS NMR spectroscopy. The temperature at which the phase transition takes place depends upon the length and the degree of unsaturation of the hydrocarbon chain. Shorter chains have less intense van der Waals interactions and undergo the transition at lower temperature. Likewise, the kinks that result from double bonds disrupt the packing and result in lower phase transition temperatures than saturated chains of comparable length. Lipids with a variety of chain lengths and degrees of unsaturation are available commercially. Further temperature and cholesterol compositions could be investigated to map out a binary composition diagram (10). For saturated lipid cholesterol mixtures, at low temperatures and cholesterol concentrations, the solid ordered (so) phase is observed. Raising the temperature above the phase transition temperature (Tm) will result in a transition to the liquid disordered (ld) state. At high cholesterol concentrations, the phase transition disappears, and only liquid ordered (lo) phase is observed, regardless of the temperature. At intermediate cholesterol concentrations, a mixture of so and lo phase is observed below Tm, and a mixture of lo and ld phases is observed above Tm. Distinguishing between so and lo phase can be difficult with MAS, so other complementary techniques may be used. For example, 2H wideline NMR spectroscopy has been used to map out lipid-cholesterol phase diagrams (11). Other nuclei that can be investigated are 31P, either with or without MAS, and 13C (12–14).


Acknowledgments


The author would like to thank the Texas A&M University students in the Physical Chemistry laboratory courses from 2007 – 2012 for their feedback on these experiments. The author gratefully acknowledges helpful discussions with Dr. Sean Bowen, Dr. Christian Hilty and Dr. Ivan Polozov in the development of these experiments.

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1H NMR MAS Investigations of Phase Behavior in Lipid Membranes

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