Article pubs.acs.org/Langmuir
Temperature-Resistant Bicelles for Structural Studies by Solid-State NMR Spectroscopy Kazutoshi Yamamoto,† Paige Pearcy,† Dong-Kuk Lee,‡ Changsu Yu,‡ Sang-Choul Im,§ Lucy Waskell,§ and Ayyalusamy Ramamoorthy*,† †
Department of Chemistry and Biophysics, University of Michigan, 930 N. University Ave., Ann Arbor, Michigan 48109-1055, United States ‡ Department of Fine Chemistry, Seoul National University of Technology, Seoul 139-743, Korea § Department of Anesthesiology, University of Michigan, VA Medical Center, Ann Arbor, Michigan 48105, United States S Supporting Information *
ABSTRACT: Three-dimensional structure determination of membrane proteins is important to fully understand their biological functions. However, obtaining a high-resolution structure has been a major challenge mainly due to the difficulties in retaining the native folding and function of membrane proteins outside of the cellular membrane environment. These challenges are acute if the protein contains a large soluble domain, as it needs bulk water unlike the transmembrane domains of an integral membrane protein. For structural studies on such proteins either by nuclear magnetic resonance (NMR) spectroscopy or X-ray crystallography, bicelles have been demonstrated to be superior to conventional micelles, yet their temperature restrictions attributed to their thermal instabilities are a major disadvantage. Here, we report an approach to overcome this drawback through searching for an optimum combination of bicellar compositions. We demonstrate that bicelles composed of 1,2-didecanoyl-sn-glycero-3-phosphocholine (DDPC) and 1,2-diheptanoyl-sn-glycero-3-phosphocholin (DHepPC), without utilizing additional stabilizing chemicals, are quite stable and are resistant to temperature variations. These temperature-resistant bicelles have a robust bicellar phase and magnetic alignment over a broad range of temperatures, between −15 and 80 °C, retain the native structure of a membrane protein, and increase the sensitivity of solid-state NMR experiments performed at low temperatures. Advantages of two-dimensional separated-local field (SLF) solid-state NMR experiments at a low temperature are demonstrated on magnetically aligned bicelles containing an electron carrier membrane protein, cytochrome b5. Morphological information on different DDPC-based bicellar compositions, varying q ratio/size, and hydration levels obtained from 31P NMR experiments in this study is also beneficial for a variety of biophysical and spectroscopic techniques, including solution NMR and magic-angle-spinning (MAS) NMR for a wide range of temperatures.
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biophysical and spectroscopic techniques.13−16 Bicelles are discoidal shaped lipid bilayers composed of a long-chain lipid and a short-chain detergent, most commonly DMPC (1,2dimyristoyl-sn-glycero-3-phosphocholine) and DHexPC (1,2dihexanoyl-sn-glycero-3-phosphocholine, also abbreviated as DHPC), with the size of the bicelle controlled by their mole ratio (known as the q ratio = [long acyl chain lipid]/[short acyl chain detergent]) and the hydration level.17 Bicelles have a similar fluidity of natural membranes and enable native folding of proteins as they contain high water content and planar lamellar phase lipid bilayer. Along with their similar physical makeup to that of natural membranes, bicelles are also optimal for various spectroscopic structural studies.12,18,19 Small bicelles with a q ratio less than 1.5 function well as a medium for
INTRODUCTION
Obtaining atomic-level structure and dynamics information is very important to completely understand the functional properties of membrane proteins.1−4 While acquiring such information has presented major challenges to most biophysical techniques, recent developments in solid-state NMR spectroscopy have enabled structural studies on membrane proteins reconstituted in model membranes.5−10 However, the utilization of near-native membrane mimetics and suitable solid-state NMR approaches to obtain physiologically relevant information from membrane proteins is limited by experimental and sample conditions such as temperature, hydration, and salt concentration.11 In fact, these difficulties severely limit the biophysical studies on membrane proteins containing large soluble domains such as single-pass or double-pass membrane proteins.12 Bicelles are widely used as biological cell membrane mimetics to study membrane protein structures by a variety of © 2015 American Chemical Society
Received: November 8, 2014 Revised: December 27, 2014 Published: January 7, 2015 1496
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Figure 1. (A) Chemical structures of long-chain DDPC lipid and short-chain DHepPC and DHexPC lipids used in the preparation of bicelles (B) in this study. times to obtain transparent solutions of DDPC/detergent bicelles. Bicelle solutions were packed in 4 mm NMR glass tubes. Preceding the NMR experiments, cooling/heating cycles between −15 and 25 °C were performed more than 5 times after inserting sample in the NMR probe in order to ensure the homogeneous magnetic alignment of bicelles. NMR Measurements. 31P solid-state NMR experiments were performed using Agilent/Varian solid-state NMR spectrometers with a 5 mm double resonance magic-angle-spinning (MAS) probe under static sample conditions with a 400 MHz NMR spectrometer and 4 mm triple resonance magic-angle-spinning probe with a 600 MHz spectrometer. 31P NMR spectra were recorded using Hahn-echo experiments with a 90° pulse length of 5 μs, an echo delay of 60 μs, and SPINAL64 proton decoupling with an RF field strength of 25 kHz.25 The 2D spectrum was obtained using 32 t1 increments with a dwell time of 78 μs, 25 ms acquisition time, 5000 scans, and a 2.5 s recycle delay. An RF field strength of 38.5 kHz was used for WIM (windowless isotropic mixing) in the t1 period of the HIMSELF sequence.
solution-state NMR spectroscopic studies, while large bicelles (with q > 2.5) are often used in solid-state NMR experiments. Amidst the breadth of applications for bicelles, their magnetic alignment is limited by the temperature range. The most frequently used bicelles consisting of DMPC:DHexPC align between a narrow range of temperatures, 30−45 °C, which is particularly problematic for heat-sensitive biomolecules.20 Since the radio-frequency (RF) pulses used for NMR experiments can heat the sample, there is considerable interest in developing heat-resistant bicelles that can align at low temperatures yet maintain the fluid lamellar phase.21−24 In this study, we report the development of novel thermally stable temperature-resistant bicelles that magnetically align over a broad range of temperatures, between −15 and 80 °C, without the need for the addition of membrane stabilizing chemicals. We experimentally demonstrate the enhancement of solid-state NMR sensitivity while maintaining the spectral resolution at low temperatures and its application for structural and dynamic studies of a single-pass membrane protein, cytochrome b5.
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RESULTS AND DISCUSSION Strategy for the Preparation of Temperature-Resistant Bicelles. The temperature range that bicelles magnetically align is dependent on the liquid-crystalline-to-gel phase transition temperature of the long-chain lipid present in bicelles as well as the various properties of short-chain detergents and long-chain lipids. In a previous study we found that using DLPC (1,2-dilauroyl-sn-glycero-3-phosphocholine, 12 carbons in acyl chains), a saturated long-chain lipid with a lower liquid-crystalline-to-gel phase transition temperature than DMPC, increased the alignment temperature range of bicelles.26,27 In this study, we systematically evaluated the combinations of long acyl chain lipids with short acyl chain detergents and found that (1) DDPC (1,2-didecanoyl-snglycero-3-phosphocholine, 10 carbons in saturated acyl chains)
EXPERIMENTAL SECTION
Materials. All phospholipids were purchased from Avanti Polar Lipids Inc (Alabaster, AL) and used without further purification. All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO). Preparation of DDPC/Detergents Bicelles. A 50 mM HEPES buffer, pH 7.5, was added to DDPC (1,2-didecanoyl-sn-glycero-3phosphocholine) powder to form hydrated DDPC liposomes. Then, a concentrated detergent solution (0.4 mg of lipid powder in 1 μL of water), i.e., DHepPC (1,2-diheptanoyl-sn-glycero-3-phosphocholine) or DHexPC (1,2-dihexanoyl-sn-glycero-3-phosphocholine) solution, was added to the DDPC liposomes. In order to control the hydration levels, an additional 50 mM HEPES buffer was added to this DDPC/ detergent solution. The resulting solution was homogeneously mixed by vortexing. Freeze-and-thaw cycles were sequentially applied five 1497
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Figure 2. (A) Proton-decoupled 31P NMR spectra of commonly used bicelles that are composed of 3.5:1 molar ratio of DMPC:DHexPC at 75% (w/ v) water hydration. (B) Models of lipid structures present at different temperatures.
phase, between 30 and 45 °C, two distinctly resolved 31P NMR resonances at −12.5 and −5.5 ppm were observed for DMPC and DHexPC, respectively. The observation of narrow 31P spectral lines indicates a high quality of magnetically aligned bicelles with the DMPC bilayer normal oriented perpendicular to the external magnetic field. The anisotropic 31P chemical shift value of the DMPC peak observed at −12.5 ppm corresponds to the scaled perpendicular frequency edge of the 31 P chemical shift anisotropy (CSA) powder pattern spectrum of an unaligned DMPC liquid-crystalline phase lipid bilayers.17,36 The 31P chemical shift of DMPC in the magnetically oriented bicelles, ωDMPC, is expressed by the equation37
with an optimized q ratio exhibits a broader range of magnetic alignment temperatures than DLPC, (2) increasing the acyl chain length of the detergent from the conventional detergent, DHexPC (or DHPC) (1,2-dihexanoyl-sn-glycero-3-phosphocholine, 6 carbons in acyl chains) to DHepPC (1,2diheptanoyl-sn-glycero-3-phosphocholine, 7 carbons in acyl chains) broadened the range of temperatures suitable for magnetic alignment, and (3) the optimization of hydration levels was crucial for magnetic alignment of DDPC bicelles. 31P NMR spectra were acquired from various combinations of DDPC and DHexPC or DHepPC to assess the morphological phases, stability, and magnetic alignment of bicelles at various temperatures, for various combinations of bicelles. The chemical structures of the molecules used in this study are shown in Figure 1A. Using these lipids, temperatureresistant bicelles were made by simply mixing detergent solutions in liposomes as shown in Figure 1B. Homogeneous bicelles can be prepared without utilizing any organic solvents in this scheme. Therefore, this straightforward and robust procedure for making temperature-resistant bicelles can be flexibly adapted for various biophysical/biochemical investigations of membrane-associated biomolecules.28 For example, prior to making these bicelles, membrane proteins can be reconstituted into either liposomes or micelles to accommodate each membrane protein purification.29 Magnetic-Alignment of Conventional DMPC:DHexPC Bicelles by 31P NMR. The characteristics and morphological structures of conventional DMPC:DHexPC bicelles have been comprehensively investigated using various biophysical techniques, such as NMR,15,30−32 electron microscopy,31,33 smallangle neutron scattering,32,34 and small-angle X-ray scattering.35 Out of these techniques, 31P NMR spectroscopy is considered to be a convenient and reliable method to monitor the magnetic alignment and the morphological phases of bicelles over a range of temperatures, as reported previously.17,36 A series of proton-decoupled 31P solid-state NMR spectra of commonly used q = 3.5 DMPC/DHexPC bicelles as a function of temperature were recorded to assess the magnetic alignment and morphological structures of bicelles as shown in Figure 2A. 31 P NMR spectra can identify the following four morphological phases: (1) a small (segregated) bicellar phase, (2) a mixed DDPC/DHexPC phase, (3) a magnetically aligned phase, and (4) a high-temperature phase.20,36 In the magnetically aligned
iso ωDMPC = ωDMPC +
1 (3 cos2 θ − 1)δ DMPCS bicelles 2
(1)
ωiso DMPC
where is the isotropic chemical shift of the DMPC bilayer. θ is the angle between the average orientation of bicelles and the static magnetic field. In this study, magnetically aligned bicelles have their average DMPC bilayer normal oriented perpendicular to the magnetic field; therefore, θ is equal to 90°. δDMPC is the average CSA of DMPC in the bilayer frame (δDMPC ≃ 30 ppm). Sbicelles is the order parameter of bicelles (Sbicelles = 0.85 at 37 °C).17 In the magnetically aligned phase, bicelles are composed of a DMPC-rich planar bilayer region and a DHexPC-rich high curvature “edge” region as shown in Figure 2B. DHexPC shortchain detergents in the edge regions partially undergo fast exchange with DMPC long-chain bilayers with increasing temperatures. This rapid miscibility of DHexPC detergents with the DMPC planar regions induced a high-field shift of the DHexPC peak at high temperatures in the magnetically aligned phase.36 Also, in the high temperature phase, when above 50 °C, a part of DHexPC detergents forms mixed micelles, which contain a small amount of DMPC and produced an isotropic peak around 0 ppm.20 The DMPC peak in this temperature range was shifted by −3 ppm due to the higher order parameter of DMPC bilayer regions.36 At lower temperatures than the bicelle temperature range, between 0 and 10 °C, the mixture of DMPC and DHexPC forms a micellar state called small segregated bicelles, which shows an isotropic 31P chemical shift around 0 ppm.20 In the temperature range of 10−20 °C, the small segregated bicelles separate into DHexPC micelles and 1498
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Figure 3. Magnetic alignment of DDPC:DHexPC bicelles. Proton-decoupled 31P NMR spectra of DDPC:DHexPC bicelles with various q (= [DDPC]/[DHexPC]) ratios (1, 2, 3, 4, and 5) recorded at the indicated temperatures with 75% (w/v) hydration level. The peaks around −8 and −12.4 ppm arise from DHexPC and DDPC, respectively. Other experimental parameters include 24 scans, 20 ms acquisition time, 4 s recycle delay, and 25 kHz SPINAL64 decoupling25 of protons. Spectra were obtained using a 400 MHz Agilent/Varian NMR solid-state NMR spectrometer with a 5 mm 1H/31P double resonance probe.
gel-phase DMPC bilayers. Around 24 °C, the gel-to-liquidcrystalline transition temperature of DMPC, a mixed DMPC/ DHexPC phase, is observed.20 These observations suggest that the morphological structures of DMPC:DHexPC bicelles are profoundly dependent on the temperature as shown in Figure 2B. In addition, the magnetically aligned q = 3.5 DMPC:DHexPC bicelles are found to be suitable for static solid-state NMR experiments for temperatures ranging only between 30 and 45 °C, which are relatively high temperatures for some heat-sensitive membrane-associated proteins such as cytochrome P450.26,38 In order to overcome the thermal instability of the magnetically aligned phase of conventional DMPC:DHexPC bicelles, in the present study, new bicelle compositions are systematically evaluated by changing the temperature and the molar q ratio. 31 P NMR Spectra of DDPC:DHexPC Bicelles Demonstrate the Use of a Low Melting Lipid To Broaden the Temperature Range of Magnetic Alignment of Bicelles. For the first optimization of the DDPC based bicelles, the most commonly used detergent DHexPC (or also abbreviated as DHPC)36 was used to examine the magnetic alignments of DDPC:DHexPC bicelles. In order to produce robust bicelles that can have a wide range of temperatures suitable for the structural studies of membrane proteins using solid-state NMR spectroscopy, proton-decoupled 31P NMR spectra of q = 1, 2, 3, 4, and 5 DDPC:DHexPC bicelles containing 75% (w/v) hydration level were recorded for temperatures between −15 and 70 °C (Figure 3). Since an unaligned phase with an ellipsoidal morphology can mainly be observed for bicelles with a large q ratio (q ≥ 6),36 bicelles with q ratio up to 5 are investigated in this study. The 31P NMR spectra shown in Figure 3 identified the four phases discussed in the previous section: a small (segregated) bicellar phase, a mixed DDPC/ DHexPC phase, a magnetically aligned phase, and a hightemperature phase.20,36 DDPC/DHexPC mixture with a q ratio of 1 exhibited an isotropic peak for all temperatures ranging from −15 to 70 °C. On the other hand, DDPC/DHexPC mixture with a q > 1 exhibited magnetically aligned bicellar phase for most temperatures as shown in Figure 3: −10 to 0, −10 to 30, −10 to 50, and −10 to 60 °C for q ratios 2, 3, 4, and 5, respectively. At −15 °C, all the bicelles with q ≥ 2 showed a mixed DDPC/DHexPC phase, which indicates that the bicelles are around the gel-to-liquid-crystalline transition temperature.
31
P NMR spectra shown in Figure 3 indicate that at higher temperatures DDPC:DHexPC combinations exist in a hightemperature phase: above 0, 40, 60, and 70 °C for q = 2, 3, 4, and 5, respectively. Chemical shift frequency of the DDPC peak observed in 31P NMR spectra of a high-temperature phase is shifted toward the low-field region. This observation indicates that a decreasing order parameter of bicelles due to the extensive miscibility of DHexPC in the planar DDPC bilayer regions and/or a fast−intermediate exchange of the partial fraction of DDPC between the planar and the rim regions of bicelles.36 In the magnetically aligned phase, 31P NMR spectra exhibited two narrow peaks, in which the ratios of intensity/ area are consistent with the q ratio. The range of temperatures for magnetic alignment increased as the q ratio increased from 2 to 5 with the q ratio of 5 presenting the widest range of alignment (between −10 and 60 °C). It is interesting to note that the temperature range of a magnetic alignment for the q = 4 DDPC:DHexPC bicelles (−10 to 50 °C) is significantly larger than that for q = 4 DLPC:DHexPC bicelles (0−40 °C).26,27 This enhancement in thermal stability of magnetically aligned DDPC:DHexPC bicelles is attributed to a lower gel-toliquid-crystalline phase transition temperature of DDPC than that of DLPC. In the magnetically aligned phase of DDPC:DHexPC bicelles (Figure 3), increasing temperature shifts the DHexPC peak toward the high-field region whereas the DDPC peak slightly shifts toward the low-field region to decrease the chemical shift difference between the two peaks due to the progressive miscibility of DHexPC from the rim regions with the planar DDPC bilayer region as explained below.32,36 Based on the “mixed bicelle model”, the experimentally observed 31P chemical shift of short-chain detergents can be described by the population of detergents in the planar bilayer regions. In this model, the long-chain lipids and the short-chain detergents are mixed in each domain of bicelles through a rapid exchange of detergents between the edge and the planar bilayer regions of bicelles.36 For q ≥ 2, where the amount of the longchain lipids (DDPC) in the edge regions is neglected, observed 31 P chemical shift of detergents, ωobs detergent, is expressed by the following equation for an asymmetric two-site chemical exchange in the fast/intermediate regime36 (k > 2.48 × 103 s−1, k is the transition probability per unit time): 1499
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Figure 4. Magnetic alignment of DDPC:DHepPC bicelles. Proton-decoupled 31P NMR spectra of DDPC:DHepPC bicelles with various q (= [DDPC]/[DHepPC]) ratios (1, 2, 3, 4, and 5) recorded at the indicated temperatures with 75% (w/v) hydration level. The peaks around −8 and −12.4 ppm arise from DHepPC and DDPC, respectively. All other experimental conditions are as given in the Figure 3 caption. edge bilayer obs ωdetergent = Πωdetergent + (1 − Π)ωdetergent
chemical shift difference between DDPC and DHepPC peaks in the magnetically aligned bicellar phase suggests progressive increments of the fractional population of DHepPC in the DDPC bilayer region, as discussed in the previous section. This enhanced exchange process is ascribed to the smaller difference in the acyl chain lengths between DDPC (10 carbons in acyl chains) and DHepPC (7 carbons in acyl chains). It has been known that the mixing of two saturated phospholipids differing in acyl chain lengths induces a lowering of the liquid-crystallineto-gel phase transition temperature of long acyl chains in lipid bilayers.39 Lowering the liquid-crystalline-to-gel phase transition temperature of planar bilayers generally enhances the stabilization of the biceller phase, as reported previously.26,27 Therefore, DDPC:DHepPC bicelles exhibit a wider temperature range of magnetic alignment phase than DDPC:DHexPC bicelles. On the contrary, DDPC:DHepPC bicelles with a q ratio of 5 do not align below −10 °C, and for temperatures above 40 °C 31P NMR peaks exhibit a significant reduction of intensities, indicating poor alignment of bicelles and/or fast− intermediate/intermediate exchange of DHepPC (and DDPC) between the rim and the planar regions (k > |(ωbilayer DHepPC − 3 −1 ωedge )/2| = 2.48 × 10 s ). These results further suggest DHepPC that DHepPC molecules, in particular, undergo nearly edge intermediate exchange (k = |ωbilayer DHepPC − ωDHepPC| ≃ 4.96 × 3 −1 10 s ) that result in the disappearance of DHepPC peaks above 40 °C (Figure 4).36 On the basis of the optimization of DDPC based bicelles, we found that DHepPC is an excellent detergent to improve the stability of bicelles that can render an increased range of temperatures for which DDPC:DHexPC bicelles magnetically align and therefore are well suited for solid-state NMR structural studies of membrane-associated proteins. Effect of Hydration on the Magnetic Alignment of DDPC:DHepPC Bicelles. Native-like dynamics of lipids in bicelles is essential to maintain a native-like bilayer environment for the structural studies of membrane-associated proteins.17,40,41 Hydration levels and temperatures substantially alter lipid dynamics as have been shown from the measurement of 1H−13C dipolar couplings and 2H quadrupole coupling parameters through solid-state NMR experiments.17,42 In this study, in order to evaluate the effect of water hydration and thermal kinetic energy, the most thermally stable DDPC:DHepPC bicelles with q = 4 were further optimized by varying the hydration level at different temperatures and
(2)
31 edge where ωbilayer detergent and ωdetergent are the P chemical shift values of detergents in planar and edge regions of bicelles, respectively. 31 In our bicellar system, ωbilayer detergent is the P chemical shift of the long-chain lipids (DDPC), −12.4 ppm, and ωedge detergent is equal to 0 ppm.36 Π is the fractional population of detergents in the planar bilayer regions.
Π=
bilayer ndetergent edge bilayer ndetergent + ndetergent
nbilayer detergent
(3)
nedge detergent
where and are the number of detergent molecules in planar and edge regions of bicelles, respectively. Therefore, an increasing fractional detergent population in the planar lipid bilayer region gives rise to a reduction in the chemical shift difference between DDPC and DHexPC peaks as seen in Figure 3. An optimization of the fast exchange process of detergent molecules in bicelles could further improve the thermal stability of magnetically aligned bicelles. Replacing DHexPC with DHepPC Broadens the Temperature of Magnetic Alignment for DDPC Based Bicelles. One of the approaches to control the miscibility of short-chain detergents with long-chain lipid bilayer of bicelles is the variation of the difference between the acyl chain lengths. In order to assess this effect and to increase the thermal stability of bicelles, proton-decoupled 31P NMR spectra of q = 1, 2, 3, 4, and 5 DDPC:DHepPC bicelles with 75% (w/v) hydration level were recorded for temperatures between −15 and 90 °C (Figure 4). As mentioned in the previous section, the spectra indicate the presence of different bicellar phases in the sample. While an isotropic phase was observed for q = 1 DDPC:DHepPC (Figure 4) like that observed for DDPC:DHexPC (Figure 3), the temperature range of magnetic alignment of DDPC:DHepPC bicelles (Figure 4) is wider than that observed for DDPC:DHexPC bicelles (Figure 3). As shown in Figure 4, magnetically aligned bicelles are present for temperatures −10 to 40, −10 to 60, −15 to 80, and −10 to 90 °C for q = 2, 3, 4, and 5 DDPC:DHepPC bicelles. The range of temperatures for magnetic alignment increases as the q ratio increased from 2 to 4 with the q ratio of 4 presenting the widest range of alignment to occur between −15 and 80 °C. This temperature range is 35 °C wider than that of DDPC:DHexPC with the q ratio of 4.39 The pronounced reduction of the 1500
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Langmuir analyzed using 31P NMR experiments (Figure 5). Our experimental results shown in Figure 5 suggest that the
Low-Temperature Solid-State NMR Experiments on Cytochrome b5 Using Temperature-Resistant Bicelles. To determine the effectiveness of the temperature-resistant bicelles for protein structural studies, we incorporated 0.67 mM U-15N-labeled cytochrome b5 into q = 4, DDPC:DHepPC bicelles containing a 5 mM Cu2+−DMPE−DTPA to speed up the spin−lattice relaxation of protons (Figure 6A). 15N chemical shift spectra were obtained using the ramped-crosspolarization (ramp-CP)46 pulse sequence (Figure 6B). Cytochrome b5 is a 16.7 kDa single-pass membrane protein
Figure 5. Effect of hydration on magnetically aligned DDPC:DHexPC bicelles. Proton-decoupled 31P NMR spectra of DDPC:DHepPC bicelles (q = 4) with hydration levels 65% (w/v), 75% (w/v), and 85% (w/v). All other experimental conditions are as given in the Figure 3 caption.
optimum hydration level of 75%(w/v) renders the widest range (−15 to 80 °C) of magnetic alignment of DDPC:DHepPC bicelles with q = 4. For hydration levels between 65% (w/v) and 85% (w/v), line broadening is observed for temperatures above 40 °C. This observation may be attributed to the high thermal kinetic energy at high temperatures that can expel water molecules from the hydration shell around the water− membrane interface, inducing dehydration of the bicellar surface. This dehydrating effect leads to a greater order of the glycerol region in the lipid head groups of bicelles,43−45 which slows the migration of detergents from the rim to the planar bilayer region of bicelles. Such a slow chemical exchange process can cause broadening of 31P NMR peaks at high temperatures.36 This fast−intermediate miscibility of shortchain detergents is pronounced at a lower hydration level, 65% (w/v), due to the lack of water molecules in the free state. In contrast, it is also known that a further increase of the hydration level, to 85% (w/v), decreases the ordering of the headgroup moieties in bicelles as previously reported.17,42 Thus, higher hydration levels progressively decrease the ordering of water molecules at the water−membrane interface, leading to a significant alteration of the local dynamics in the headgroup regions of bilayers.17,42 Consequently, 31P NMR spectra of DDPC:DHepPC bicelles with 85% (w/v) hydration exhibits a narrow range of magnetic alignment temperatures. Therefore, the stability of bicelles requires an optimal hydration level, which is a fine balance between the hydrogen-bonding network around the membrane surface and the thermal kinetic energy. In summary, 4:1 DDPC:DHepPC bicelles with 75% (w/v) hydration level are the optimum condition to obtain magnetically aligned temperature-resistant bicelles in our current study. This systematic optimization of bicellar components and conditions resulted in temperature-resistant bicelles that provide the most suitable of environment for the solid-state NMR studies of membrane proteins as demonstrated in the following section.
Figure 6. Magnetically aligned bicelles enable low-temperature solidstate NMR experiments on cytochrome b5. (A) Representation of cytochrome b5 embedded in magnetically aligned bicelles. (B) 15N NMR spectra of DDPC:DHepPC bicelles (4.0, 75% (w/v) hydration) containing a uniformly 15N-labeled cytochrome b5 obtained using the ramped-cross-polarization (CP)46 pulse sequence at the indicated temperatures. As reported previously, peaks appearing in the 60−90 ppm chemical shift range arise from the amide-NH groups present in the relatively immobile transmembrane domain (residues 105−127) of cytochrome b5, whereas the peaks appearing between 100 and 140 ppm originate from the highly mobile soluble domain of cytochrome b5.12 Spectra obtained at −10 °C (black) and 20 °C (red) are overlapped for an easy comparison in (B). (C) Proton-decoupled 31P NMR spectra confirm the magnetic-alignment of DDPC:DHepPC bicelles containing cytochrome b5 for a temperature range of −10 to 40 °C as indicated. The peaks appearing approximately −12.4 and −8 ppm are from DDPC lipids and DHepPC detergents. The isotropic peak around 0 ppm arises from a phosphate buffer in the sample. As the temperature decreases, the chemical shift separation between the lipid and detergent 31P peaks increases, indicating a decrease in the mixing of detergent molecules with lipids. Spectra were obtained with a 600 MHz Agilent/Varian solid-state NMR spectrometer using a 4 mm triple resonance E-free MAS probe. 15N ramped-CP spectra (B) were obtained using 0.5 ms contact time, 25 ms acquisition time, 50 kHz SPINAL64 decoupling25 of protons, 4000 scans, and a 2.5 s recycle delay. Proton-decoupled 31P NMR spectra (C) were obtained from 24 scans, 20 ms acquisition time, and a 4 s recycle delay. 1501
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Langmuir located in the endoplasmic reticulum of liver cells. This hemecontaining electron carrier protein alters the enzymatic activities of cytochrome P450s, which metabolize ∼75% of pharmaceutical compounds in current use. It has been demonstrated that the transmembrane α-helix of cytochrome b5 is essential for its functional roles. Therefore, atomic-level structural and dynamic studies of the full-length membranebound cytochrome b5 are crucial to understand its function.12 31 P NMR spectra shown in Figure 6C indicate that the bicelle/ protein complex (with a 1:500 (mol/mol) protein:lipid ratio) is well aligned at various temperatures. An isotropic peak from the phosphate buffer appears at 0 ppm. For the purpose of the sensitivity enhancement of low γ (like 15N) nuclei in membrane proteins, a small amount of Cu2+−DMPE−DTPA was added in bicelles as reported in the literature.24 This paramagneticcopper-chelated lipid shortens the spin−lattice (T1) relaxation time due to paramagnetic relaxation enhancements (PRE) and therefore enables a fast NMR signal acquisition without altering spectral resolution as demonstrated previously.47−49 15 N NMR spectra were obtained to evaluate the sensitivity of NMR signals from magnetically aligned bicelles containing cytochrome b5 for various temperatures (Figure 6A). The amino acid residues in the three domains of cytochrome b5 resonate in distinct chemical shift regions of the spectrum: the soluble domain (100−135 ppm), the linker domain, and the transmembrane domain (70−100 ppm).12 As the sample temperature is decreased to −10 °C, the signal intensity is enhanced for both the soluble and transmembrane domains due to the motional restriction of the protein at low temperatures, allowing for an improved CP efficiency. The increments of the sensitivity enhancement from 20 to −10 °C, as extracted from Figure 6B, are 1.54 times (119.3 ppm), 1.69 times (116.7 ppm), 1.29 times (110.9 ppm), 1.23 times (80.9 ppm), 1.51 times (72.1 ppm), and 3.47 times (32.2 ppm) as shown in Supporting Information Figure S1. The peak associated with the lysine side chain (32.2 ppm) becomes clearly visible as the temperature of the sample decreases. These experimental results demonstrate the sensitivity enhancement rendered by the temperature-resistant DDPC:DHepPC bicelles at low temperatures, which can be exploited in multidimensional solid-state NMR studies on heatsensitive membrane proteins. 2D SLF Experiments on Magnetically Aligned DMPC/ DHexPC Bicelles Containing a Uniformly 15N-Labeled Cytochrome b5. A 2D separated local field (SLF), HIMSELF (heteronuclear isotropic mixing leading to spin exchange via the local field),50,51 which correlates the 15N chemical shift with 1 H−15N dipolar coupling, spectrum was obtained (Figure 7). In this 2D spectrum, peaks from the transmembrane lysine and arginine side chains are observed with a high intensity (70−80 ppm). Further, the simulation of the helical span of resonances in the 2D spectrum indicates that the transmembrane helix is ∼17 ± 3° tilted away from the lipid bilayer normal. While the observed wheel pattern of resonances does not fit perfectly to the simulated wheel of an ideal α-helix, the tilt angle of ∼17 ± 3° is comparable to our previous observations: ∼17 ± 3° and 15 ± 3° from DLPC (12 carbons in acyl chains) and DMPC (14 carbons in acyl chains), respectively.12,26 Simulated helical wheels for different tilt angles are shown in Figure S3 of the Supporting Information. The distortion of resonances from the simulated circular pattern in the SLF spectrum indicates that the Pro 116 residue induces a kink in the transmembrane αhelix region.12 This characteristic of a kinked transmembrane α-
Figure 7. 2D HIMSELF solid-state NMR experiments on magnetically aligned bicelles reveal the topology of the transmembrane domain of cytochrome b5. (A) 2D separated local field (SLF) spectrum, correlating 15N chemical shift with 1H−15N dipolar coupling, obtained using the HIMSELF (heteronuclear isotropic mixing leading to spin exchange via the local field) pulse sequence50,51 and magnetically aligned DDPC:DHepPC bicelles (q = [DDPC]/[DHepPC] = 4.0, 75% (w/v) hydration) containing a 0.67 mM uniformly 15N-labeled cytochrome b5 and 5 mM Cu2+−DMPE−DTPA at 0 °C. (B) A structural model depicting the tilt of the transmembrane helix relative to the lipid bilayer normal and a kink induced by the Pro-116 residue.
helix aids in overcoming the hydrophobic mismatch to maintain the transmembrane topology, irrespective of the lipid bilayer thickness.52
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CONCLUSIONS In summary, we have developed a novel combination of lipid (DDPC) and detergent (DHepPC) to create bicelles that magnetically align for a wide range of temperatures including low temperatures than any of the previously reported bicellar systems. Not only do these bicelles allow for the structural analysis of membrane proteins in near native membrane bilayer conditions, even at low temperatures to avoid heating effects, they also render enhanced spectral sensitivity. The straightforward procedure of preparing thermally stable bicelles without the use of organic solvents may also be useful to various studies of membrane associated biomolecules by a variety of biophysical techniques.53−55 The variation of bicelle compositions has been shown as an effective method for broadening and lowering magnetic alignment temperatures for various biophysical techniques including NMR spectroscopy.21−23,26,27 As demonstrated in this study, several parameters and compositions can be used to optimize the stability of bicelles. In this study, commonly used zwitterionic phosphatidylcholines (PCs), which are the most abundant lipids in biomembranes, were utilized. The choice of a model bicellar membrane is not limited to PCs; further improvement of the stability of temperature-resistant bicelles could be achieved through the following approaches: (1) Stabilizer chemical additives, such as cholesterol22,23 and cholesterol-3-sulfate,21,27 for ordering the acyl chains of longchain lipids or decreasing the phase transition temperatures. (2) Lower phase transition temperature lipids.26,27 (3) Optimum detergents,27,32 which have most favorable length/size of acyl chains or a charged headgroup to induce the ideal “rapid” miscibility with the planar regions for the stabilization of bicelles; for example, it is known that a cholate-based detergent, 3-([3-cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate (CHAPSO), can be also used for stabilizing the rim regime of bicelles, since CHAPSO has a large facial amphiphilicity via β-face of CHAPSO structure.32 (4) Polar aprotic solvents, which allow the ordering of lipid head groups and prevent ice formation, such as dimethyl sulfoxide 1502
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Langmuir (DMSO).56 DMSO can be fully dissolved in water, be distributed throughout the membrane−water interface, and order both the polar region and a part of the hydrophobic acyl chains region of lipid bilayers.56 (5) The use of charged chemical additives, such as hexadecyltrimethylammonium bromide (CTAB), to control the surface charge of bicelles could stabilize bicelle formation.27 (6) Inclusion of small amounts of salts, such as sodium chloride or potassium chloride, could improve and stabilize the magnetic alignment.33 The combination of temperature-resistant bicelles reported in this study with these approaches could greatly improve the stability of bicelles. Such studies are currently under continual investigation in our lab. While there is considerable interest in utilizing lowtemperature experiments, such as dynamic nuclear polarization (DNP) solid-state MAS, to fully exploit the sensitivity enhancement, poor spectral resolution due to frozen molecular motions and/or conformational heterogeneity pose tremendous difficulties. Such difficulties could partly be overcome by the use of our temperature-resistant bicelles that enable experiments at temperatures as low as −15 °C. In fact, the use of Overhauser DNP experiments as demonstrated on aligned lipid bilayers at ambient temperature in a very recent study60 would significantly benefit from the use of temperatureresistant bicelles to study membrane proteins. In addition, the use of temperature-resistant bicelles with the paramagnetic relaxation enhancement-assisted data collection at low temperatures24,47−49 will likely overcome some of the difficulties related to the poor sensitivity of NMR techniques.
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(4) Van den Berg, B.; Clemons, W. M., Jr.; Collinson, I.; Modis, Y.; Hartmann, E.; Harrison, S. C.; Rapoport, T. A. X-ray structure of a protein-conducting channel. Nature 2004, 427, 36−44. (5) Han, Y.; Hou, G.; Suiter, C. L.; Ahn, J.; Byeon, I. J.; Lipton, A. S.; Burton, S.; Hung, I.; Gor’kov, P. L.; Gan, Z.; Brey, W.; Rice, D.; Gronenborn, A. M.; Polenova, T. Magic angle spinning NMR reveals sequence-dependent structural plasticity, dynamics, and the spacer peptide 1 conformation in HIV-1 capsid protein assemblies. J. Am. Chem. Soc. 2013, 135, 17793−17803. (6) Wang, S.; Munro, R. A.; Shi, L.; Kawamura, I.; Okitsu, T.; Wada, A.; Kim, S. Y.; Jung, K. H.; Brown, L. S.; Ladizhansky, V. Solid-state NMR spectroscopy structure determination of a lipid-embedded heptahelical membrane protein. Nat. Methods 2013, 10, 1007−1012. (7) Gustavsson, M.; Verardi, R.; Mullen, D. G.; Mote, K. R.; Traaseth, N. J.; Gopinath, T.; Veglia, G. Allosteric regulation of SERCA by phosphorylation-mediated conformational shift of phospholamban. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 17338− 17343. (8) Yang, J.; Aslimovska, L.; Glaubitz, C. Molecular dynamics of green proteorhodopsin in lipid bilayers by solid-state NMR. J. Am. Chem. Soc. 2011, 133, 4874−4881. (9) Radoicic, J.; Lu, G. J.; Opella, S. J. NMR structures of membrane proteins in phospholipid bilayers. Q. Rev. Biophys. 2014, 47, 249−283. (10) Eddy, M. T.; Ong, T. C.; Clark, L.; Teijido, O.; van der Wel, P. C.; Garces, R.; Wagner, G.; Rostovtseva, T. K.; Griffin, R. G. Lipid dynamics and protein-lipid interactions in 2D crystals formed with the β-barrel integral membrane protein VDAC1. J. Am. Chem. Soc. 2012, 134, 6375−6387. (11) Xu, J.; Dürr, U. H.; Im, S. C.; Gan, Z.; Waskell, L.; Ramamoorthy, A. Bicelle-enabled structural studies on a membraneassociated cytochrome b5 by solid-state MAS NMR spectroscopy. Angew. Chem., Int. Ed. 2008, 47, 7864−7867. (12) Dü rr, U. H.; Yamamoto, K.; Im, S. C.; Waskell, L.; Ramamoorthy, A. Solid-state NMR reveals structural and dynamical properties of a membrane-anchored electron-carrier protein, cytochrome b5. J. Am. Chem. Soc. 2007, 129, 6670−6671. (13) Dürr, U. H.; Gildenberg, M.; Ramamoorthy, A. The magic of bicelles lights up membrane protein structure. Chem. Rev. 2012, 112, 6054−6074. (14) Prosser, R. S.; Evanics, F.; Kitevski, J. L.; Al-Abdul-Wahid, M. S. Current applications of bicelles in NMR studies of membraneassociated amphiphiles and proteins. Biochemistry 2006, 45, 8453− 8465. (15) Sanders, C. R.; Hare, B. J.; Howard, K. P.; Prestegard, J. H. Magnetically-oriented phospholipid micelles as a tool for the study of membrane-associated molecules. Prog. Nucl. Magn. Reson. Spectrosc. 1994, 26, 421−444. (16) Marcotte, I.; Auger, M. Bicelles as model membranes for solid and solution-state NMR studies of membrane peptides and proteins. Concepts Magn. Reson. 2005, 24A, 17−37. (17) Yamamoto, K.; Soong, R.; Ramamoorthy, A. Comprehensive analysis of lipid dynamics variation with lipid composition and hydration of bicelles using nuclear magnetic resonance (NMR) spectroscopy. Langmuir 2009, 25, 7010−7018. (18) Dvinskikh, S. V.; Dürr, U. H. N.; Yamamoto, K.; Ramamoorthy, A. High-resolution 2D NMR spectroscopy of bicelles to measure the membrane interaction of ligands. J. Am. Chem. Soc. 2007, 129, 794− 802. (19) Dvinskikh, S. V.; Dürr, U.; Yamamoto, K.; Ramamoorthy, A. A high-Resolution solid-state NMR approach for the structural studies of bicelles. J. Am. Chem. Soc. 2006, 128, 6326−6327. (20) Sternin, E.; Nizza, D.; Gawrisch, K. Temperature dependence of DMPC/DHPC mixing in a bicellar solution and its structural implications. Langmuir 2001, 17, 2610−2616. (21) Shapiro, R. A.; Brindley, A. J.; Martin, R. W. Thermal stabilization of DMPC/DHPC bicelles by addition of cholesterol sulfate. J. Am. Chem. Soc. 2010, 132, 11406−11407.
ASSOCIATED CONTENT
S Supporting Information *
Procedure to prepare bicelles, amino acid sequence of cytochrome b5, and 2D HIMSELF spectra at different temperatures. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected] (A.R.). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research is supported by funds from NIH (GM084018 and GM095640 to A.R.) and partly by a VA Merit Review grant to L.W.
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