Article pubs.acs.org/Langmuir
Bicelles Exhibiting Magnetic Alignment for a Broader Range of Temperatures: A Solid-State NMR Study Kazutoshi Yamamoto, Paige Pearcy, and Ayyalusamy Ramamoorthy* Biophysics and Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109-1055, United States ABSTRACT: Bicelles are increasingly used as model membranes to suitably mimic the biological cell membrane for biophysical and biochemical studies by a variety of techniques including NMR and X-ray crystallography. Recent NMR studies have successfully utilized bicelles for atomicresolution structural and dynamic studies of antimicrobial peptides, amyloid peptides, and membrane-bound proteins. Though bicelles composed with several different types of lipids and detergents have been reported, the NMR requirement of magnetic alignment of bicelles limits the temperature range in which they can be used and subsequently their composition. Because of this restriction, low-temperature experiments desirable for heat-sensitive membrane proteins have not been conducted because bicelles could not be aligned. In this study, we characterize the magnetic alignment of bicelles with various compositions for a broad range of temperatures using 31P static NMR spectroscopy in search of temperature-resistant bicelles. Our systematic investigation identified a temperature range of magnetic alignment for bicelles composed of 4:1 DLPC:DHexPC, 4:1:0.2 DLPC:DHexPC:cholesterol, 4:1:0.13 DLPC:DHexPC:CTAB, 4:1:0.13:0.2 DLPC:DHexPC:CTAB:cholesterol, and 4:1:0.4 DLPC:DHexPC:cholesterol-3-sulfate. The amount of cholesterol-3-sulfate used was based on mole percent and was varied in order to determine the optimal amount. Our results indicate that the presence of 75 wt % or more water is essential to achieve maximum magnetic alignment, while the presence of cholesterol and cholesterol-3-sulfate stabilizes the alignment at extreme temperatures and the positively charged CTAB avoids the mixing of bicelles. We believe that the use of magnetically aligned 4:1:0.4 DLPC:DHexPC:cholesterol-3-sulfate bicelles at as low as −15 °C would pave avenues to study the structure, dynamics, and membrane orientation of heat-sensitive proteins such as cytochrome P450 and could also be useful to investigate protein−protein interactions in a membrane environment.
■
INTRODUCTION High-resolution studies on the structure, dynamics, aggregation, and orientation of membrane-bound peptides and proteins are essential to better understand their biological functions.1−4 While using the native biological cell membrane is not feasible for most studies, model membranes such as bicelles, micelles, liposomes, multilamellar vesicles (MLVs), and nanodisks are commonly utilized.5−8 These model membranes are successful because either their structure and composition closely mimic the natural cell membrane or they enable the development of new methods such as cutting-edge solid-state NMR techniques.9−15 Though each of these model membranes offers unique advantages, recent studies have shown that bicelles are more suitable for a variety of biochemical and biophysical applications. Bicelles are composed of long chain lipids and short chain detergents, and the size and phase transition of properties depend on the lipid:detergent ratio.16−20 The main advantages of bicelles include the presence of high water content and planar lamellar phase lipid bilayer, which allow for a similar fluidity to that of natural membranes.21−23 In addition to their cell membrane similarities, the magnetic alignment of large bicelles enables the easy measurement of structural and © 2014 American Chemical Society
geometrical constraints from the embedded proteins by solidstate NMR experiments.24−28 Rapidly tumbling isotropic bicelles, with the q ratio up to 1, are frequently used in highresolution structural studies of membrane proteins by solution NMR. Use of bicelles as an alignment medium to measure residual dipolar couplings (RDCs) from water-soluble proteins by solution NMR spectroscopy and to crystallize membrane proteins for X-ray studies are some of their unique advantages.29,30 The intrinsic curvature of toroidal pores in bicelles has been used to study the role of curvature on the membrane disruption by antimicrobial peptide MSI-7831 and IAPP (Islet Amyloid Polypeptide Protein) amyloid32 peptides. The main limitation of bicellesparticularly for solid-state NMR applicationsis the restriction on the type of membrane components that can be used to form bicelles.33,34 Since a widespread application of bicelles depends on the variation of the lipid membrane composition as all proteins and peptides do not function in the same membranes, there is considerable interest in developing bicelles with various membrane Received: November 13, 2013 Revised: January 11, 2014 Published: January 24, 2014 1622
dx.doi.org/10.1021/la404331t | Langmuir 2014, 30, 1622−1629
Langmuir
Article
Because of these reasons, we examined the magnetic alignment of bicelles with various compositions for a broad range of temperatures. Phosphorus-31 NMR spectra of 4:1 DLPC:DHexPC, 4:1:0.2 DLPC:DHexPC:cholesterol, 4:1:0.13 DLPC:DHexPC:CTAB, 4:1:0.13:0.2 DLPC:DHexPC:CTAB:cholesterol, and 4:1:0.4 DLPC:DHexPC:cholesterol-3-sulfate are used to examine the magnetic alignment of bicelles; chemical structures of these molecules can be found in Figure 1. We demonstrate that the use of cholesterol and cholesterol3-sulfate, and lipids with lower phase transition temperatures, broadens the range of temperatures in which bicelles can be magnetically aligned.
compositions. We refer the readers to the recent comprehensive review articles for further details on bicelles.16−19,34 The presence of bulk water in bicelles has been shown to be useful in enabling native-like folding of membrane proteins containing large soluble domains like cytochrome b 5 , cytochrome P450, and cytochrome P450 reductase and could be useful to study other single- and double-pass membrane proteins.35,36 These proteins have been proven to be extremely difficult to crystallize in their full-length membrane-bound form as it is difficult to offer an environment that would enable native-like folding for both the transmembrane and soluble domains. On the other hand, a combined use of solid-state NMR studies on large bicelles and solution NMR studies on isotropic bicelles has successfully rendered high-resolution structural studies on cytochrome b5 and cytochromes b5−P450 complex.3,34 While there are many significant unique advantages with bicelles, the main limitation for NMR studies is the requirement of the magnetic alignment of bicelles. Bicelles reported so far in the literature do not align at very low or high temperatures.34,37−41 As a result, NMR studies using bicelles are commonly carried out in a narrow range of temperatures which may not be suitable to carry out cutting-edge multidimensional NMR experiments in the functional forms of the protein or peptide. For example, the most commonly used DMPC:DHexPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine:1,2-dicaproyl-sn-glycero-3-phosphocholine) bicelles magnetically align between 32 and 40 °C.37,42,43 However, a recent study showed that doping standard DMPC:DHexPC bicelles with cholesterol-3-sulfate broadens the range of alignment temperatures to about 25−45 °C.37 As outlined in Table 1, the
■
Table 1. A Summary of the Temperature Range of Magnetic Alignment for Various Bicelle Compositions Measured from Solid-State NMR Experiments Presented in This Study composition of bicelles
molar ratio
alignment temp (°C)
DMPC/DHexPC DLPC/DHexPC DLPC/DHexPC/cholesterol DLPC/DHexPC/cholesterol-3-sulfate DLPC/DHexPC/CTAB DLPC/DHexPC/cholesterol/CTAB
3.5:1 4:1 4:1:0.2 4:1:0.4 4:1:0.13 4:1:0.13:0.2
30−45 4−50 3−90 −15 to 90 5−60 2−90
MATERIALS AND METHODS
Materials. All phospholipids were purchased from Avanti Polar Lipids Inc. (Alabaster, AL). Cholesterol, cholesterol-3-sulfate, CTAB, and organic solvents were purchased from Sigma-Aldrich (St. Louis, MO). All these chemicals were used without further purification. Preparation of Bicelles. 18.7 mg of DLPC (1,2-dilauroyl-snglycero-3-phosphatidylcholine) and 3.4 mg of DHexPC (1,2dihexanoyl-sn-glycero-3-phosphatidylcholine) corresponding to a molar ratio, q = [DLPC]/[DHexPC], of 4 were cosolubilized in chloroform. Solvent was removed under a stream of N2 gas to produce a lipid film on the walls of a glass vessel, which was kept in vacuum overnight to remove residual solvent. 16.3 μL of 50 mM phosphate buffer, pH 7.5, with 20% glycerol content was added to lipids. The resulting mixture of extreme viscosity was homogenized by vortexing and four freeze/heat cycles between liquid nitrogen and 40 °C. Bicelles with other compositions were also prepared using a procedure very similar to this. A homogeneously mixed sample was packed in a 4 mm NMR glass tube and used for subsequent NMR experiments. Prior to NMR measurements, the sample inside the probe was treated with a minimum of five cooling/heating cycles between 0 and 25 °C to ensure homogeneous magnetic alignment of bicelles. This procedure was used to prepare bicelles with various compositions investigated in this study. Phosphorus-31 NMR Experiments. All 31P NMR spectra were obtained from a 400 MHz Varian solid-state NMR spectrometer using a 4 mm 1H/X double-resonance electric-field-free BioMAS probe. A Hahn-echo pulse sequence (90°−tau−180°−tau−acquisition; tau = 50 μs) with a 25 kHz radio-frequency proton decoupling was used. All 31P spectra are referenced to phosphoric acid at 0 ppm.
■
RESULTS AND DISCUSSION The strategy employed in this study was to explore the effects of using lower phase transition temperature lipids, varied acyl chain length detergents, and the addition of membrane stabilizing components (cholesterol, CTAB, and cholesterol3-sulfate) on the alignment of bicelles in order to develop bicelles which align over a broad range of temperatures. We systematically studied the effects of each of these variants through manipulating only one element and then combining those we found most favorable in the end to develop an optimal bicelle. Our results are described in the following sections. 31 P NMR Spectra of 4:1 DMPC:DHexPC vs Temperature. To evaluate the magnetic alignment of bicelles over a range of temperatures, proton-decoupled 31P NMR spectra can be used as reported in previous studies. 31P NMR spectra of the most commonly used bicelles, composed of 3.5:1 molar ratio of DMPC:DHexPC at 75% (w/v) water hydration, were recorded at different temperatures to determine the magnetic alignment temperature range. A bicellar phase, composed of DMPC bilayer surrounded by a rim of DHexPC (having a complex morphology that depends on the bicelle composition), spontaneously align in the presence of magnetic field with
limits of bicelle alignment continue to be pushed34,37,39,40 with the discovery of bicelle compositions that align as low as 8 °C.41 However, oftentimes such low alignment temperatures can only be achieved in the presence of artificial lipids whose structures are significantly different than what would be found in a biological membrane. Therefore, there is considerable interest in developing bicelles with near-native mimetic components that align at a significantly lower temperature to avoid sample heating and to retain the stable form of the protein for a long NMR data acquisition. Such bicelles, for example, would enable multidimensional solid-state NMR structural studies on heat-sensitive membrane proteins26 and also could utilize rapid data collection approaches.14 In addition, lowering the sample temperature could slow down the dynamics of the soluble domain of single-pass and doublepass membrane proteinsa significant advantage in enhancing the sensitivity and resolution of solid-state NMR experiments. 1623
dx.doi.org/10.1021/la404331t | Langmuir 2014, 30, 1622−1629
Langmuir
Article
Figure 1. Molecular structures of lipids used in the preparation of bicelles. DLPC, DHepPC, DHexPC, cholesterol, hexadecyltrimethylammonium bromide (CTAB), and cholesterol-3-sulfate.
Figure 2. (A) Proton-decoupled 31P NMR spectra of most 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.
magnetic alignment of bicelles as indicated by the broad peak at 10 ppm for the DMPC gel-phase bilayers and isotropic peak for the DHexPC micelles, and thus the components are no longer aligned. These observations suggest that samples containing well-aligned bicelles between 30 and 45 °C are suitable for static solid-state NMR experiments, but outside of that range, the bicelles are not adequate. In the alignment temperature range from 0 to 10 °C DMPC and DHexPC are present as micelles, but once heated to 15 °C the micelles separate to form individual DHexPC micelles and gel-phase DMPC bilayers. A mixed DMPC/DHexPC phase is indicated at temperatures near the gel-to-liquid-crystalline point, 24 °C. Above the bicelle temperature range, ∼45 °C, DMPC and DHexPC mix in a monolayer. The chain order of DMPC decreases while the chain order of DHexPC increases. At much higher temperatures than the bicelle temperature range, DHexPC forms a micelle and subsequently dissolves a small amount of DMPC.44
the DMPC bilayer normal perpendicular to the external magnetic field direction. Because of the magnetic alignment of bicelles, narrow spectral lines are obtained from DMPC and DHexPC. The 31P NMR spectra observed between 30 and 45 °C (Figure 2A) consist of narrow peaks for DMPC and DHexPC with 3.5:1 intensity/area ratios in agreement with the 3.5:1 molar ratio of DMPC:DHexPC in bicelles. These spectra indicate that the bicelles are well aligned. The peaks observed at 0 ppm arise from the isotropic lipid phase, whereas the peaks observed in the high-field region are from aligned bicelles. Both DMPC and DHexPC peaks shift toward the high-field region, and the chemical shift difference between them decreases with the increasing temperature, which indicates decreasing alignment. This chemical shift difference and DHexPC intensity decreased considerably at 50 and 60 °C, and the isotropic peak appears at 0 ppm, indicating the formation of DHexPC mixed micelles. Decreasing the temperature below the liquid crystalline to gel phase transition of DMPC abolishes the 1624
dx.doi.org/10.1021/la404331t | Langmuir 2014, 30, 1622−1629
Langmuir
Article
Figure 3. Proton-decoupled 31P NMR spectra of bicelles composed of (A) 4:1 DLPC:DHexPC, (B) 4:1:0.2 DLPC:DHexPC:cholesterol, (C) 4:1:0.13 DLPC:DHexPC:CTAB, and (D) 4:1:0.13:0.2 DLPC:DHexPC:CTAB:cholesterol at the indicated temperatures and 90% (w/v) water hydration.
Figure 4. Proton-decoupled 31P NMR spectra of 4:1 DLPC:DHexPC bicelles with varying amounts of cholesterol-3-sulfate (1, 5, 9, 13, 17, and 21 mol % given as A, B, C, D, E, and F, respectively) at the indicated temperatures and with 75% (w/v) water hydration. A sharp peak from the phosphate buffer is observed at 4 ppm in all spectra. Isotropic peaks observed in 31P spectra, which were obtained at high temperatures, are indicated by dotted vertical lines.
Figure 2B shows models of lipid structures present at different temperatures. DLPC-Containing Bicelles Align at Lower Temperatures Than DMPC-Containing Bicelles. Modifications to the most commonly used bicelles have shown to change the temperature range of alignment. Proton-decoupled 31P NMR spectra of 4:1 DLPC:DHexPC, 4:1:0.2 DLPC:DHexPC:cholesterol, 4:1:0.13 DLPC:DHexPC:CTAB, and 4:1:0.13:0.2
DLPC:DHexPC:CTAB:cholesterol bicelles at various temperatures and 90% (w/v) water hydration were recorded and are shown in Figure 3 to determine if these different combinations of detergents and lipids could produce more optimal temperature ranges for magnetic alignment and subsequent NMR studies with biological molecules. The ratio of DLPC used was based on the initial systematic investigation of q ratios of long chain lipids and subsequent alignment done by 1625
dx.doi.org/10.1021/la404331t | Langmuir 2014, 30, 1622−1629
Langmuir
Article
Warschawski et al.42 The observed results suggest that the DLPC:DHexPC bicelles were magnetically aligned very well between 4 and 50 °C and are suitable for solid-state NMR studies as shown in Figure 3A. The 31P NMR spectra observed for temperatures above 50 °C and even at 0 °C also indicate some degree of alignment but may not be ideally suited for solid-state NMR experimental studies. Our results agree with a previous study, where it was found that 3-(cholamidopropyl)dimethylammonio-2-hydroxyl-1-propanesulfonate (CHAPSO):DLPC bicelles align over a broader range of temperatures than the commonly used DMPC:DHexPC;39 however instead of using CHAPSO, we used DHexPC. Taking both our study and the previous study into account, results corroborate that DLPC has a wider range of temperatures for alignment than DMPC. The observed decrease in the chemical shift difference between DLPC and DHexPC with the increasing sample temperature is to some extent similar to that of DMPC:DHexPC bicelles explained above. Nevertheless, when compared to the conventionally used DMPC:DHexPC bicelles, the DLPC:DHexPC: bicelles magnetically align even at lower temperatures. This may be because of the lower liquid crystalline to gel phase transition of DLPC (−2 °C) than that of DMPC (24 °C). We also note that a previous study demonstrated the use of DLPC bicelles to study peptides.45 Our recent study35 showed that DLPC:DHexPC bicelles are useful to conduct structural studies on a ∼57 kDa cytochrome P450 using 2D HIMSELF (heteronuclear isotropic mixing leading to spin exchange via the local field) or HERSELF (heteronuclear rotating-frame leading to spin exchange via the local field)46 solid-state NMR spectroscopy. Effects of Cholesterol-3-sulfate on Bicelles. Cholesterol is known to incorporate into lipid bilayers, increase the order of acyl chains of lipids, and broaden the main phase transition temperature of lipids. We wanted to exploit the effect of cholesterol to broaden the temperature range for which bicelles can be aligned, particularly to align bicelles at low temperatures. A previous study found that cholesterol-3-sulfate, when added to DMPC:DHexPC bicelles, allowed for lower than room temperature alignment.38 In this study, we incorporated cholesterol into bicelles composed of DLPC and were able to align bicelles at temperatures much lower than room temperature. As shown in Figure 3B, the addition of cholesterol to DLPC:DHexPC bicelles enhances the intensity of aligned peaks, from DLPC in particular, indicating a reduction of any disorder in the alignment of bicelles for a broad range of temperatures. This could be attributed to the increase in the rigidity of acyl chains of DLPC and broadening its phase transition temperature due to the presence of cholesterol. Proton-decoupled 31P NMR spectra of DLPC:DHexPC bicelles with varying amounts of cholesterol-3-sulfate (1, 5, 9, 13, 17, and 21 mol %) at the indicated temperatures and with 75% (w/v) water hydration were collected as shown in Figure 4. The presence of cholesterol-3-sulfate enabled the magnetic alignment of bicelles at low temperatures. Even in the presence of a small amount of cholesterol-3-sulfate, bicelles align at −5 °C, while alignment at −10 °C was observed when a high concentration of cholesterol-3-sulfate was used. Other observations are similar to that observed for DLPC:DHexPC bicelles without cholesterol-3-sulfate. Thus, increasing the amount of cholesterol-3-sulfate allows bicelles to align at lower temperatures that would be suitable for solid-state NMR studies.
Effects of CTAB on Bicelles. In this study, we have investigated the effect of CTAB on DLPC and DMPC containing bicelles. Our results show that the addition of CTAB enhances the intensities of 31P peaks of both DLPC and DHexPC but broadens the DMPC peak below 10 °C as shown in Figure 3C. In addition, though the chemical shift difference between DLPC and DHexPC observed from DLPC:DHexPC:CTAB bicelles decreased with the increasing temperature, it is larger when compared to DLPC:DHexPC bicelles without CTAB. This observation suggests that the presence of the positively charged CTAB reduces the mixing of DHexPC in the lamellar phase DLPC lipid bilayers. But, the presence of CTAB introduces phase separation above ∼60 °C, resulting in the formation of DHexPC mixed micelles as indicated by the appearance of an isotropic peak at 0 ppm. Overall, the DLPC:DHexPC:CTAB bicelles are most notably well aligned at higher temperatures than the commonly used bicelles and could be useful for solid-state NMR studies between ∼5 and ∼60 °C. Interestingly, it has previously been shown that the addition of a positively charged CTAB increases the alignment of bicelles at higher temperatures,47 however not with these combinations of lipids, which have a different range than what has been previously reported. Optimizing the Effects of Cholesterol and CTAB on Bicelles. After the preceding experiments were conducted, we decided to use both cholesterol and CTAB in bicelles to employ both of their effects on the model membrane. As shown in Figure 3D, the presence of both cholesterol and CTAB retained the features observed in Figures 3B,C. In particular, the spectra observed at high temperatures are similar to that of DLPC:DHexPC:CTAB but without the isotropic peak. Thus, DLPC:DHexPC:CTAB:cholesterol bicelles exhibit excellent alignment for temperatures ranging from ∼2 to ∼90 °C. This more optimal temperature range than the more typical DMPC:DHexPC bicelles allows for a wider range of NMR studies using solution NMR experiments at higher temperatures and solid-state NMR at lower temperatures. Furthermore, these bicelles containing a suitable amount of cholesterol and CTAB offer a more realistic membrane environment for proteins and other biological molecules that are active at specific temperatures. Higher Hydration Levels Enable Magnetic Alignment of Bicelles at Low Temperatures. Changing the hydration level of bicelles was our next variant explored. Protondecoupled 31P NMR spectra of DLPC:DHexPC bicelles with and without 10 mol % cholesterol-3-sulfate at the indicated temperatures and with 90% (w/v) water hydration, a higher hydration level than was previously tested, were collected and are shown in Figure 5. The observations are very similar to that in Figure 4, but a higher amount of hydration improved the alignment for a broad range of temperatures. It is important to note that magnetic-alignment of bicelles with the higher hydration level can be seen even at −15 °C, though the lines are broader due to some disorder in the alignment. Our results reveal that the chemical shift frequency of aligned DLPC in both these bicelles does not change significantly unlike that in less hydrated bicelles. This observation suggests that these bicelles can be used for studies at high temperatures. For example, these bicelles could be utilized as alignment media in the measurement of residual dipolar couplings from watersoluble proteins using solution NMR experiments. Effect of Varying the Acyl Chain Length of the Detergent on Bicelles. It was previously deduced that as the 1626
dx.doi.org/10.1021/la404331t | Langmuir 2014, 30, 1622−1629
Langmuir
Article
Figure 6. Proton-decoupled 31P NMR spectra of bicelles composed of (A) 4:1 DLPC:DHexPC and (B) 4:1 DLPC:DHepPC at the indicated temperatures and with 75% (w/v) water hydration.
Figure 5. Proton-decoupled 31P NMR spectra of 4:1 DLPC:DHexPC bicelles without (A) and with 10 mol % cholesterol-3-sulfate (B) at the indicated temperatures and with 90% (w/v) water hydration.
of their lipid components. Finding a suitable combination of components to form bicelles that remain in the bicellular phase, without being too fluid or presenting as micelles, is key to increasing magnetic-alignment and developing bicelles appropriate for analysis at a wide range of temperatures. We have demonstrated that by changing the bicelle composition, the alignment temperature range could be expanded due to different liquid crystalline to gel phase transition temperatures and altered mixing of long chain lipids and short chain detergents. In order for success with our experiments it was important to test the various conditions for bicelles, such as hydration and detergent with different acyl chain length individually. Our results suggest that the use of a suitable amount of cholesterol and CTAB with a low-melting lipid-like DLPC result in fluid lamellar phase bicelles suitable for NMR studies at a wide range of temperatures like −15 to 60 °C. It would be fruitful to characterize these bicelle mixtures using other biophysical techniques like TEM and dynamic light scattering to further understand the size of the aggregates at different temperatures. Since the inclusion of a ligand, peptide, or protein into a lipid bilayer can alter the main phase transition temperature of the lipid, often it is not easy to prepare the lamellar phase bilayers. The use of bicelles containing a suitable amount of cholesterol and CTAB could overcome this difficulty. With this study one can better devise the composition of bicelles for structural studies on peptides and proteins. Applications of bicellesthat align at very low-temperatures developed in this studyto investigate the structure of heat-sensitive membrane proteins by solid-state NMR spectroscopy are in progress in our laboratory. The use of low-temperature bicelles could also reduce the recycle delay to enable fast NMR data collection by combining with approaches to shorten the spin−lattice relaxation times by using a paramagnetic metal-chelated lipid14,48 or by doping with paramagnetic metal ions in other forms as demonstrated elsewhere.49−53 Bicelles can also be used in the structural
difference in carbon chain length between the detergent and lipids decreases, the miscibility of the two components increases. With this increased miscibility, the alignment temperature range was found to increase.32 Therefore, we thought an investigation of the effect of increasing the detergent chain length on DLPC containing bicelles could be useful as DLPC has already proven to have a wider temperature range of alignment and the previous study did not analyze DLPC bicelles. Proton-decoupled 31P NMR spectra of DLPC:DHexPC and DLPC:DHepPC bicelles at the indicated temperatures and with 75% (w/v) water hydration are shown in Figure 6. Replacing the DHexPC (6 carbon) detergent with a longer acyl-chain containing detergent, DHepPC (7 carbon), significantly changed the alignment properties of bicelles. As seen in Figure 6, the chemical shift difference between the lipid and detergent 31P peaks rapidly decreases with the increasing temperature. The two peaks coalesce around ∼50 °C, a higher temperature than that which DHexPC and DLPC 31P peaks combine, indicating that the DHepPC easily mixes with the DLPC lipid. Interestingly, the chemical shift frequency of aligned DLPC in DLPC:DHepPC bicelles does not change significantly unlike that observed for DLPC:DHexPC bicelles. These results suggest that even with 75% water hydration DLPC:DHepPC bicelles can be used for studies at higher temperatures. Should lower water content bicelles be desired, as in MAS solid-state NMR studies on intrinsic membrane proteins, DLPC:DHepPC bicelles could be utilized.
■
CONCLUSIONS The primary purpose of this study was to develop bicelles with a broader range of temperature alignments to aid with NMR spectroscopic studies of proteins and biological molecules. This was achieved through changing the composition of bicelles and measuring proton-decoupled 31P NMR spectra of bicelles. Bicelles align in an external magnetic field based on the phase 1627
dx.doi.org/10.1021/la404331t | Langmuir 2014, 30, 1622−1629
Langmuir
Article
(14) Yamamoto, K.; Xu, J.; Kawulka, K. E.; Vederas, J. C.; Ramamoorthy, A. Use of a copper-chelated lipid speeds up NMR measurements from membrane proteins. J. Am. Chem. Soc. 2010, 132, 6929−6931. (15) Wang, T.; Park, Y. B.; Caporini, M. A.; Rosay, M.; Zhong, L.; Cosgrove, D. J.; Hong, M. Sensitivity-enhanced solid-state NMR detection of expansin’s target in plant cell walls. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 16444−16449. (16) Kim, H. J.; Howell, S. C.; Van Horn, W. D.; Jeon, Y. H.; Sanders, C. R. Recent advances in the application of solution NMR spectroscopy to multi-span integral membrane proteins. Prog. Nucl. Magn. Reson. Spectrosc. 2009, 55, 335−360. (17) 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. (18) Marcotte, I.; Auger, M. Bicelles as model membranes for solidand solution-state NMR studies of membrane peptides and proteins. Concepts Magn. Reson. 2005, 24A, 17−37. (19) Warschawski, D. E.; Arnold, A. A.; Beaugrand, M.; Gravel, A.; Chartrand, É.; Marcotte, I. Choosing membrane mimetics for NMR structural studies of transmembrane proteins. Biochim. Biophys. Acta 2011, 1808, 1957−1974. (20) Opella, S. J.; Marassi, F. M. Structure determination of membrane proteins by NMR spectroscopy. Chem. Rev. 2004, 104, 3587−3606. (21) Whiles, J. A.; Deems, R.; Vold, R. R.; Dennis, E. A. Bicelles in structure-function studies of membrane-associated proteins. Bioorg. Chem. 2002, 30, 431−442. (22) 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. (23) Cardon, T. B.; Dave, P. C.; Lorigan, G. A. Magnetically aligned phospholipid bilayers with large q ratios stabilize magnetic alignment with high order in the gel and L (alpha) phases. Langmuir 2005, 21, 4291−4298. (24) Dvinskikh, S. V.; Yamamoto, K.; Dürr, U. H.; Ramamoorthy, A. Sensitivity and resolution enhancement in solid-state NMR spectroscopy of bicelles. J. Magn. Reson. 2007, 184, 228−235. (25) Howard, K. P.; Opella, S. J. High-resolution solid-state NMR spectra of integral membrane proteins reconstituted into magnetically oriented phospholipid bilayers. J. Magn. Reson. 1996, 112, 91−94. (26) Dvinskikh, S. V.; Dürr, U. H.; 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. (27) 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. (28) Soong, R.; Smith, P. E.; Xu, J.; Yamamoto, K.; Im, S. C.; Waskell, L.; Ramamoorthy, A. Proton-evolved local-field solid-state NMR studies of cytochrome b5 embedded in bicelles, revealing both structural and dynamical information. J. Am. Chem. Soc. 2010, 132, 5779−5788. (29) Tjandra, N.; Bax, A. Direct measurement of distance and angles in biomolecules by NMR in a dilute liquid crystalline medium. Science 1997, 278, 1111−1114. (30) Fischer, M. W.; Losonczi, J. A.; Weaver, J. L.; Prestegard, J. H. Domain orientation and dynamics in multidomain proteins from residual dipolar couplings. Biochemistry 1999, 38, 9013−9022. (31) Dvinskikh, S.; 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. (32) Smith, P. E.; Brender, J. R.; Ramamoorthy, A. Induction of negative curvature as a mechanism of cell toxicity by amyloidogenic peptides: the case of islet amyloid polypeptide. J. Am. Chem. Soc. 2009, 131, 4470−4478.
studies of membrane proteins using magic angle spinning solidstate NMR techniques as demonstrated previously.54−58 The temperature-resistance bicelles developed in this study could be valuable for studies carried out under ultrafast MAS conditions.59,60
■
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected] (A.R.). Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS This research was supported by funds from the NIH (GM084018 and GM095640 to A.R.).
■
REFERENCES
(1) 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. (2) Comellas, G.; Rienstra, C. M. Protein structure determination by magic-angle spinning solid-state NMR, and insights into the formation, structure, and stability of amyloid fibrils. Annu. Rev. Biophys. 2013, 42, 515−536. (3) Ahuja, S.; Jahr, N.; Im, S. C.; Vivekanandan, S.; Popovych, N.; Le Clair, S. V.; Huang, R.; Soong.; Xu., R.; Yamamoto, K.; Nanga, R. P.; Bridges, A.; Waskell, L.; Ramamoorthy, A. A model of the membranebound cytochrome b5-cytochrome P450 complex from NMR and mutagenesis data. J. Biol. Chem. 2013, 288, 22080−22095. (4) Verardi, R.; Shi, L.; Traaseth, N. J.; Walsh, N.; Veglia, G. Structural topology of phospholamban pentamer in lipid bilayers by a hybrid solution and solid-state NMR method. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 9101−9106. (5) Byeon, I. J.; Hou, G.; Han, Y.; Suiter, C. L.; Ahn, J.; Jung, J.; Byeon, C. H.; Gronenborn, A. M.; Polenova, T. Motions on the millisecond time scale and multiple conformations of HIV-1 capsid protein: implications for structural polymorphism of CA assemblies. J. Am. Chem. Soc. 2012, 134, 6455−6466. (6) Hagn, F.; Etzkorn, M.; Raschle, T.; Wagner, G. Optimized phospholipid bilayer nanodiscs facilitate high-resolution structure determination of membrane proteins. J. Am. Chem. Soc. 2013, 135, 1919−1925. (7) Kijac, A. Z.; Li, Y.; Sligar, S. G.; Rienstra, C. M. Magic-angle spinning solid-state NMR spectroscopy of nanodisc-embedded human CYP3A4. Biochemistry. 2007, 46, 13696−13703. (8) Sugawara, M.; Resende, J. M.; Moraes, C. M.; Marquette, A.; Chich, J. F.; Metz-Boutigue, M. H.; Bechinger, B. Membrane structure and interactions of human catestatin by multidimensional solution and solid-state NMR spectroscopy. FASEB J. 2010, 24, 1737−1746. (9) Bhate, M. P.; McDermott, A. E. Protonation state of E71 in KcsA and its role for channel collapse and inactivation. Proc. Natil. Acad. Sci. U. S. A. 2012, 109, 15265−15270. (10) Cady, S. D.; Schmidt-Rohr, K.; Wang, J.; Soto, C. S.; Degrado, W. F.; Hong, M. Structure of the amantadine binding site of influenza M2 proton channels in lipid bilayers. Nature 2010, 463, 689−692. (11) Mote, K. R.; Gopinath, T.; Veglia, G. Determination of structural topology of a membrane protein in lipid bilayers using polarization optimized experiments (POE) for static and MAS solid state NMR spectroscopy. J. Biomol. NMR 2013, 57, 91−102. (12) Tang, W.; Knox, R. W.; Nevzorov, A. A. A spectroscopic assignment technique for membrane proteins reconstituted in magnetically aligned bicelles. J. Biomol. NMR 2012, 54, 307−316. (13) Dave, P. C.; Inbaraj, J. J.; Lorigan, G. A. Electron paramagnetic resonance studies of magnetically aligned phospholipid bilayers utilizing a phospholipid spin label. Langmuir 2004, 20, 5801−58108. 1628
dx.doi.org/10.1021/la404331t | Langmuir 2014, 30, 1622−1629
Langmuir
Article
(33) De Angelis, A. A.; Opella, S. J. Bicelle samples for solid-state NMR of membrane proteins. Nat. Protoc. 2007, 2, 2332−2338. (34) Dürr, U. H.; Gildenberg, M.; Ramamoorthy, A. The magic of bicelles lights up membrane protein structure. Chem. Rev. 2012, 112, 6054−6074. (35) Yamamoto, K.; Gildenberg, M.; Ahuja, S.; Im, S. C.; Pearcy, P.; Waskell, L.; Ramamoorthy, A. Probing the transmembrane structure and topology of microsomal cytochrome-p450 by solid-state NMR on temperature-resistant bicelles. Sci. Rep. 2013, 3, 2556. (36) Yamamoto, K.; Dürr, U. H.; Xu, J.; Im, S. C.; Waskell, L.; Ramamoorthy, A. Dynamic interaction between membrane-bound fulllength cytochrome P450 and cytochrome b5 observed by solid-state NMR spectroscopy. Sci. Rep. 2013, 3, 2538. (37) 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. (38) Brindley, A. J.; Martin, R. W. Effect of divalent cations on DMPC/DHPC bicelle formation and alignment. Langmuir 2012, 28, 7788−7796. (39) Wang, H.; Eberstadt, M.; Olejniczak, E. T.; Meadows, R. P.; Fesik, S. W. A liquid crystalline medium for measuring residual dipolar couplings over a wide range of temperatures. J. Biomol. NMR 1998, 12, 443−446. (40) Cho, H. S.; Dominick, J. L.; Spence, M. M. Lipid domains in bicelles containing unsaturated lipids and cholesterol. J. Phys. Chem. B 2010, 114, 9238−9245. (41) Cho, G.; Fung, B. M.; Reddy, V. B. Phospholipid bicelles with positive anisotropy of the magnetic susceptibility. J. Am. Chem. Soc. 2001, 123, 1537−1538. (42) Triba, M. N.; Devaux, P. F.; Warschawski, D. R. Effects of lipid chain length and unsaturation on bicelle stability. A phosphorus NMR study. Biophys. J. 2006, 91, 1357−1367. (43) Sanders, C. R.; Schwonek, J. P. Characterization of magnetically orientable bilayers in mixtures of dihexanoylphosphatidylcholine and dimyristoylphosphatidylcholine by solid-state NMR. Biochemistry 1992, 31, 8898−8905. (44) 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. (45) Whiles, J. A.; Glover, K. J.; Vold, R. R.; Komives, E. A. Methods for studying transmembrane peptides in bicelles: consequences of hydrophobic mismatch and peptide sequence. J. Magn. Reson. 2002, 158, 149−156. (46) Dvinskikh, S. V.; Yamamoto, K.; Ramamoorthy, A. Heteronuclear isotropic mixing separated local field NMR spectroscopy. J. Chem. Phys. 2006, 125, 34507. (47) Losonczi, J. A.; Prestegard, J. H. Improved dilute bicelle solutions for high-resolution NMR of biological macromolecules. J. Biomol. NMR 1998, 12, 447−451. (48) Yamamoto, K.; Vivekanandan, S.; Ramamoorthy, A. Fast NMR data acquisition from bicelles containing a membrane-associated peptide at natural-abundance. J. Phys. Chem. B 2011, 115, 12448−55. (49) Tang, M.; Berthold, D. A.; Rienstra, C. M. Solid-State NMR of a large membrane protein by paramagnetic relaxation enhancement. J. Phys. Chem. Lett. 2011, 2, 1836−1841. (50) Nadaud, P. S.; Sengupta, I.; Helmus, J. J.; Jaroniec, C. P. Evaluation of the influence of intermolecular electron-nucleus couplings and intrinsic metal binding sites on the measurement of 15 N longitudinal paramagnetic relaxation enhancements in proteins by solid-state NMR. J. Biomol. NMR 2011, 51, 293−302. (51) Wickramasinghe, N. P.; Ishii, Y. Sensitivity enhancement, assignment, and distance measurement in C-13 solid-state NMR spectroscopy for paramagnetic systems under fast magic angle spinning. J. Magn. Reson. 2006, 181, 233−243. (52) Linser, R.; Chevelkov, V.; Diehl, A.; Reif, B. Sensitivity enhancement using paramagnetic relaxation in MAS solid-state NMR of perdeuterated proteins. J. Magn. Reson. 2007, 189, 209−216. (53) Knight, M. J.; Pell, A. J.; Bertini, I.; Felli, I. C.; Gonnelli, L.; Pierattelli, R.; Herrmann, T.; Emsley, L.; Pintacuda, G. Structure and
backbone dynamics of a microcrystalline metalloprotein by solid-state NMR. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 11095−11100. (54) 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. (55) Tian, F.; Losonczi, J. A.; Fischer, M. W.; Prestegard, J. H. Sign determination of dipolar couplings in field-oriented bicelles by variable angle sample spinning (VASS). J. Biomol. NMR 1999, 15, 145−150. (56) Lancelot, N.; Elbayed, K.; Piotto, M. Applications of variableangle sample spinning experiments to the measurement of scaled residual dipolar couplings and 15N CSA in soluble proteins. J. Biomol. NMR 2005, 33, 153−161. (57) Lancelot, N.; Elbayed, K.; Piotto, M. Measurement of scaled residual dipolar couplings in proteins using variable-angle sample spinning. J. Biomol. NMR 2004, 29, 259−269. (58) Kishore, A. I.; Prestegard, J. H. Molecular orientation and conformation of phosphatidylinositides in membrane mimetics using variable angle sample spinning (VASS) NMR. Biophys. J. 2003, 85, 3848−3857. (59) Zhou, D. H.; Nieuwkoop, A. J.; Berthold, D. A.; Comellas, G.; Sperling, L. J.; Tang, M.; Shah, G. J.; Brea, E. J.; Lemkau, L. R.; Rienstra, C. M. Solid-state NMR analysis of membrane proteins and protein aggregates by proton detected spectroscopy. J. Biomol. NMR 2012, 54, 291−305. (60) Asami, S.; Reif, B. Proton-detected solid-state NMR spectroscopy of aliphatic sites: application to crystalline systems. Acc. Chem. Res. 2013, 46, 2089−97.
1629
dx.doi.org/10.1021/la404331t | Langmuir 2014, 30, 1622−1629
