Phase Transitions in Small Isotropic Bicelles - Langmuir (ACS

Feb 27, 2018 - Isotropic phospholipid bicelles are one of the most prospective membrane mimetics for the structural studies of membrane proteins in so...
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Article Cite This: Langmuir 2018, 34, 3426−3437

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Phase Transitions in Small Isotropic Bicelles Erik F. Kot,†,‡ Sergey A. Goncharuk,†,§ Alexander S. Arseniev,†,‡ and Konstantin S. Mineev*,†,‡ †

Shemyakin−Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences RAS, str. Miklukho-Maklaya 16/10, Moscow 117997, Russian Federation ‡ Moscow Institute of Physics and Technology, Institutsky per., 9, 141700 Dolgoprudnyi, Russian Federation § Lomonosov Moscow State University, Leninskiye Gory, 1, Moscow 119991, Russian Federation S Supporting Information *

ABSTRACT: Isotropic phospholipid bicelles are one of the most prospective membrane mimetics for the structural studies of membrane proteins in solution. Recent works provided an almost full set of data regarding the properties of isotropic bicelles; however, one major aspect of their behavior is still under consideration: the possible mixing between the lipid and detergent in the bilayer area. This problem may be resolved by studying the lipid phase transitions in bicelle particles. In the present work, we investigate two effects: phase transitions of bilayer lipids and temperature-induced growth of isotropic bicelles using the NMR spectroscopy. We propose an approach to study the phase transitions in isotropic bicelles based on the properties of 31P NMR spectra of bilayer-forming lipids. We show that phase transitions in small bicelles are “fractional”, particles with the liquid-crystalline and gel bilayers coexist in solution at certain temperatures. We study the effects of lipid fatty chain type and demonstrate that the behavior of various lipids in bilayers is reproduced in the isotropic bicelles. We show that the temperature-induced growth of isotropic bicelles is not related directly to the phase transition but is the result of the reversible fusion of bicelle particles. In accordance with our data, rim detergents also have an impact on phase transitions: detergents that resist the temperature-induced growth provide the narrowest and most expressed transitions at higher temperatures. We demonstrate clearly that phase transitions take place even in the smallest bicelles that are applicable for structural studies of membrane proteins by solution NMR spectroscopy. This last finding, together with other data draws a thick line under the long-lasting argument about the relevance of small isotropic bicelles. We show with certainty that the small bicelles can reproduce the most fundamental property of lipid membranes: the ability to undergo phase transition.

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INTRODUCTION Bicelles are membrane mimetics that are formed in the mixtures of lipids and specific detergents, such as the bile salt derivatives1 and short-chain phospholipids.2−4 Bicelles are known to form the discoidal particles,5 with the lipids forming a patch of planar bilayer and detergents associating into the “rim” of the disc. The size of the particles is controlled by the ratio between the lipid and rim-forming detergent (q) in quite a wide range, starting from 20 kDa. Large bicelles (q > 2−3 for various mixtures) were shown to orient spontaneously in the strong magnetic field and are used in solid-state NMR spectroscopy to prepare the oriented bilayer samples6 and in solution to create the anisotropic environment for the soluble proteins and measure the residual dipolar couplings.7 Due to the specified ability, such large particles are referred to as “anisotropic” bicelles. In contrast, solutions with small particles which are not capable of spontaneous orientation are called isotropic bicelles (IsoBs). IsoBs have several features that make them an almost perfect membrane mimetic for structural studies of membrane proteins.8−10 Unlike detergent micelles, bicelles contain lipids © 2018 American Chemical Society

that form the plane patch of membrane and properly mimic some properties of the lipid bilayer.11,12 The lipid composition of bicelles can be varied to simulate the membranes of different cells or their microdomains.13 Cholesterol, sphingolipids, glycerolipids, lipids with unsaturated fatty chains, or anionic headgroups can be added to the isotropic bicelles.14−18 IsoBs can be formed using the very mild rim-forming surfactants, such as Facades,19 that do not cause the unfolding of soluble globular domains of large membrane proteins.20 Unlike lipid−protein nanodiscs, IsoBs are cheaper and easier to prepare and the size of bicelles is lower, which makes them compatible with solution NMR spectroscopy. They allow the exchange of matter between the particles in solution, which is convenient for the studies of protein−protein interactions. IsoBs have found their application in X-ray crystallography;21 however, the widest use Received: October 17, 2017 Revised: February 12, 2018 Published: February 27, 2018 3426

DOI: 10.1021/acs.langmuir.7b03610 Langmuir 2018, 34, 3426−3437

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choline (POPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC), 3-[(3cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), Facade-EPC and Facade-EM), and sodium dodecyl sulfate (SDS) were the product of Avanti Polar Lipids Inc. D2O was the product of Cambridge Isotope Laboratory. Sample Preparation. Here and below q* denotes the effective lipid/detergent ratio, taking into account the presence of the detergent in the monomeric form.12 To prepare the bicelles, an ∼10 mg of lipids in a dry powder were dissolved by the necessary amount of 10% stock solution of CHAPS, DHPC, Facade-EPC, or Facade-EM and then the total volume of the sample was adjusted to 500 μL by the addition of 30 mM NaPi buffer, pH 7.0, containing 10% of D2O. The resulting mixture was frozen and then heated to 40−50 °C in the ultrasonic bath for 3−4 times. To vary the q ratio, the solution was either diluted by the stock solution of detergent or added to the necessary amount of lipid in dry powder and put into the ultrasonic bath for 5−6 min at 40 °C. For the dilution experiments, 10% w/w solution of bicelles at necessary q was prepared. To vary the concentration of bicelles, necessary portions of the initial solution were diluted by the 3.0−5.0 mM solution of CHAPS, depending on the bilayer lipid and q* of bicelles. To prepare the lipid−protein nanodiscs (LPNs) samples, necessary amounts of MSP1D1 were added to the mixture of DMPC and sodium cholate (1:1) dissolved in the ND-buffer (20 mM Tris pH 8.0, 100 mM NaCl) and incubated for 1 h. Total lipid/MSP ratio was adjusted to 65. To assemble LPNs, the Biobeads SM-2 (Biorad, USA) (1 g per 70 mg of detergent) were added, and the suspension was shaken at room temperature for 12−16 h.26 The resulting solution was filtered (to remove Biobeads), centrifuged at 14000g for 20 min, and then the supernatant was purified by the size-exclusion chromatography and concentrated to 500 μl. 31 P NMR Experiments. 31P NMR spectra were recorded on the Bruker Avance 600 MHz spectrometer equipped with the broadband double resonance probe. A single pulse experiment was employed using the π/6 pulse of 4 μs, a relaxation delay of 3 s, and an acquisition time of 2.7 s with broadband WALTZ16 proton decoupling at the field strength of 1.42 kHz. A preacquisition delay of 10 min was used before each experiment to ensure the equilibrium state of the system. Spectra were referenced indirectly with respect to the proton signal of trimethylsilylpropanoic acid (TSP) at 0.0 ppm using the gyromagnetic ratio. To determine the chemical shifts and to measure the fractions of gel/liquid-crystalline bicelles, the 31P spectra were deconvoluted using the Lorentzian lineshapes and nonlinear regression in Wolfram Mathematica software. The temperature was varied in either direction to exclude the hysteresis and control the equilibrium state of the sample. 31 P diffusion experiments were performed using the double stimulated echo experiment,27 designed to suppress the effects of convection, with diffusion delay equal to 500 ms and encoding gradient equal to 5 ms, with the gradient pulse strength being varied linearly from 1 to 52 G/cm in 32 iterations. The pulse sequence included the broadband WALTZ16 proton decoupling at the field strength of 1.42 kHz both during the relaxation delay and acquisition. Summary duration of the diffusion experiment was equal to 8 h. The data analysis included the simultaneous deconvolution of two DPPC peaks with Lorentzian lineshapes and serial processing of the whole data set with the common peak positions and line widths in Wolfram Mathematica software, to increase the reliability of approximation. Six repetitions were made at each temperature/q* and paired Student test was used to measure the reliability of the changes (p-values). 1 H NMR Experiments. All diffusion 1H NMR spectra were recorded on the Bruker Avance 700 spectrometer with the working frequency of protons equal to 700 MHz, equipped with the roomtemperature triple resonance TXI probe. Diffusion was measured using the double stimulated echo pulse sequence with the efficient suppression of convection effects and of the intense solvent signal.28 Diffusion experiments were recorded with 32 scans and 32 increments of the encoding gradient pulse (gradient pulse strength was varied linearly from 12 to 52 G/cm). Summary length of the encoding

of this membrane mimetic was established in solution NMR studies of membrane proteins.8 Recent works provided an almost full set of data regarding the behavior of IsoBs. Size and concentration of detergents in monomeric form were determined for many types of bicelles as a function of the lipid/detergent ratio and temperature.5,12,22 This resulted in the ideal bicelle models that fit the experimental data and describe the shape of bicelles, assuming the absence of lipid/detergent mixing.5,12,23 However, one major aspect of IsoBs behavior is still under consideration: the possible mixing between the lipids and detergents in the planar area or rim of the particles. In accordance with the recent studies, small particles, applicable for the structural studies by solution NMR spectroscopy, (radius less than 3.5 nm) and large IsoBs (greater than 3.5 nm) behave similarly below the phase transition temperature of the bilayer-forming lipid and follow the ideal bicelle models. However, the dramatic temperature-induced growth is observed for the large bicelles.12,22 Above some critical temperature, which is close to the phase transition temperature of bilayer-forming lipid, large IsoBs begin to increase their radius. This growth is too strong to be explained by the change of lipid packing properties that occur during the phase transition and does not seem to stop at any particular temperature. This effect depends on the type of membrane lipid and rim-forming agent. Thus, some researchers postulate that the small IsoBs are characterized by the mixing between the lipid and detergent since they do not demonstrate a temperature-induced growth and refer to the results of computer simulations,22,24 and others assume that the large IsoBs are subject to the mixing at high temperatures.12 In our previous work, we put forward a hypothesis that the state of lipids in small bicelles is the same as observed in large bicelles below the phase transition temperature.12 The indirect data, in particular, the analysis of the protein−lipid contacts in the DMPC/CHAPS and DMPC/DHPC mixtures, do not reveal any presence of the detergent in close proximity to the membrane protein in small IsoBs regardless of the temperature and size of the bicelles.11,12 On the other hand, the saturation transfer experiments with POPC/DHPC bicelles show that at least the transient contacts are observed between the DHPC methyl groups and terminal residues of transmembrane helices in such a mixture, while the lipid−protein contacts are prevailing for the buried part of the membrane domains.25 One of the possible ways to investigate the lipid/detergent mixing and presence of lipid bilayer in IsoBs is to study the lipid phase transitions and state of lipids in bicelles of various size and compositions. Phase transitions in the isotropic DMPC/ DHPC bicelles were already probed using the infrared spectroscopy, and the study revealed that such transitions do take place in q = 0.75 and q = 1.0 mixtures but seem to be absent in q = 0.5 particles.24 However, it is a single IsoB system, and the data obtained for q = 0.5 bicelles do not allow one to draw the definitive conclusion about the absence or presence of the phase transition. Studies of phase transitions in various systems with different temperature-dependent behavior are required. In the present work, we investigate two effects in a variety of systems: the phase transitions and temperatureinduced growth of IsoBs using the 31P NMR spectroscopy and 1 H NMR diffusion measurements.

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EXPERIMENTAL SECTION

Materials. Lipids and detergents (1,2-dimyristoyl-sn-glycero-3phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho3427

DOI: 10.1021/acs.langmuir.7b03610 Langmuir 2018, 34, 3426−3437

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Figure 1. 31P NMR may be used to detect the lipid phase transitions in small bicelles. 31P NMR spectra of DPPC/CHAPS bicelles prepared with q* = 1.5, 1.0, and 0.8 recorded at 323 (blue), 303 (red), 293 (green), and 283 (magenta) K. Peaks corresponding to liquid-crystalline and gel lipids are indicated by * and **, respectively.

Figure 2. Effect of q*. (A) Chemical shift temperature dependencies recorded for narrow and broad peaks observed in 31P spectra of DPPC/CHAPS bicelles, which were prepared with various q* (see the legend). Average linear approximation of temperature dependencies is plotted by the dashed (liquid-crystalline lipids) and dotted (gel lipids) lines. For q = 0.6, the peak, corresponding to gel lipids, is too broad to get the chemical shifts with necessary precision. (B) The fraction of the gel phase DPPC/CHAPS bicelles (relative intensity of the broad peak in 31P spectra) is plotted as a function of temperature for various q*. gradient pulse was 4.0 ms, duration of the diffusion delay was 300−400 ms. The gradients were calibrated using the diffusion coefficient of residual water in 100% D2O at 25 °C. Then at least a 30 min interval was used after the change of the temperature to ensure the equilibrium state of the system. To minimize the effects of signal overlap and magnetic field inhomogeneity, the integral of the most intense and narrow peak, corresponding to N-(CH3)n moieties, was used to determine the diffusion coefficients of DMPC and CHAPS. This approach appeared to be the most error-resistant in comparison to

averaging of several peaks of the compounds. Additionally, use of the most narrow peak allows the more adequate averaging of the diffusion coefficients between the particles of various size. Data Analysis. To analyze the diffusion coefficients and convert them to the radii of particles, we used the formalism, described in detail in our previous work.12 The analysis included the corrections for the diffusion obstruction due to the high concentration of particles and due to the discoidal shape of bicelles.29,30 Experimental errors were determined using the Monte Carlo analysis. 3428

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Figure 3. Radii of DPPC/CHAPS q* = 1.50 and 1.35 bicelles measured for the (A) narrow and (B) broad peaks in 31P NMR spectrum at 298 and 293 K based on the NMR diffusion coefficients. p-Values were calculated using the Student test.

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RESULTS AND DISCUSSION Detection of Phase Transitions in IsoBs. 31P NMR is actively used to study the state of lipids in membrane mimetics, however, mostly for the systems with large particles. One of the most common applications of 31P is monitoring of the orientation of large bicelles in strong magnetic field.31,32 This process is temperature-dependent, and, at some point, the isotropic chemical shift of phosphorus (close to 0 ppm) becomes anisotropic (shifted by 10−20 ppm), which indicates the orientation of particles. 31P spectra were also utilized to measure the concentration of the monomeric detergent in IsoBs.5,24 Thus, we expected to observe the changes in 31P chemical shifts or line widths upon the lipid phase transition or as a manifestation of mixing between the lipid and detergents in either the rim or planar area of IsoBs. We first tested the DPPC/CHAPS system: the size of these bicelles was determined for the wide range of q*,12 and phase transition of DPPC occurs at a convenient temperature of 41 °C, allowing the detection of NMR spectra 30−40° below and above the critical temperature. The first experiments were performed at q* = 1.5; under these conditions particles with the radius of 5.1 nm (at 35 °C) are formed and expressed temperature-induced growth of bicelles is observed.12 At high temperatures, the single and narrow peak is observed in the 1D 31P NMR spectra of IsoBs solution at −0.8 ppm (Figure 1, left panel). Surprisingly, below the phase transition, this single peak becomes split into the narrow peak and wide peak, which is shifted upfield. The populations of two peaks depend on the ambient temperature: intensity of the wide peak increases and that of the narrow peak decreases at lower temperatures until only the single wide peak is left in the NMR spectra at 10 °C. The temperature coefficients of the 31P chemical shift for the narrow peak are the same above and below the critical temperature and are equal to 2.5 ppb/K. Temperature coefficient for the wide peak is much higher and is equal to 8.5 ppb/K (Figure 2A). Thus, we can make two conclusions. First of all, 31P NMR can be used to monitor the lipid phase transitions in IsoBs (at least for DPPC): it is manifested in NMR spectra by the presence of wide, upfield shifted and broad peak with the relatively high temperature coefficient of chemical shift. Second, the phase transition in isotropic bicelles is “fractional”. At some temperatures gel and liquid-crystalline lipids coexist in solution. This may be interpreted in two ways: either some lipids are in liquid or gel phases within the same bicelle or one fraction of

bicelles is in the gel phase and the other fraction is liquidcrystalline. Two situations could be distinguished clearly by measuring the diffusion coefficients for broad and narrow peaks in NMR spectra. 31 P NMR Diffusion Experiments. We recorded the 31P DOSY spectra of q* = 1.5 DPPC/CHAPS bicelles at 25 °C and treated separately the signals in NMR spectra (Figure S1). The experiment revealed the substantially different diffusion coefficients for the two peaks, which supports that the broad and narrow signals in 31P spectra correspond to the different types of particles in solution: liquid and gel-phase bicelles. Bicelles in the fluid phase had radii equal to 4.00 ± 0.08 nm, while the diffusion of gel-phase bicelles can be described with the radius of 5.82 ± 0.11 nm (Figure 3). This is counterintuitive because the melting of bicelles should result in the increase of radius according to the ideal bicelle model,12 due to the increased area per lipid headgroup, observed in liquid bilayers in comparison to the gel bilayers.33 However, we observe the opposite, the size of fluid-phase bicelles is decreased substantially. This can be considered as evidence in favor of the mixing between the lipid and detergent in liquidcrystalline bicelles. If liquid-crystalline and gel phase bicelles have different morphology and structure, this could result in the presence of two subsets of particles with various extent of mixing between the lipid and detergent. In addition, such a mixing could explain the reduced phase transition temperature of DPPC in bicelles, compared to the bilayer membrane. However, we suppose that it is unlikely for several reasons. When the whole pool of particles is measured by 1H diffusion, the radius of liquid-crystalline bicelles above the phase transition is greater than the radius of bicelles in the gel phase. At 40 °C radius of DPPC/CHAPS q* = 1.5 bicelles equals 6.9 ± 0.2 nm. This is greater than the size of IsoBs at 35 °C, the starting point of phase transition (5.1 ± 0.1 nm) and is greater than the radius of gel phase bicelles at 25 °C. Thus, it is not a general property of bicelles in the liquid-crystalline phase to be smaller than gel-phase particles, the increased size of gel bicelles is observed only during the “fractional” phase transition. Therefore, this effect cannot be explained by the different morphology and lipid/detergent mixing in two kinds of particles. Additionally, the migration of detergents to the bilayer area of bicelles should not decrease but rather increase the size of particles because such mixing is equivalent to the growth of the effective q ratio. 3429

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Figure 4. Effect of the lipid chain length and saturation. (A) 31P NMR spectra of DMPC/CHAPS q* = 1.5 (left panel) and POPC/CHAPS q* = 1.2 (right panel) bicelles recorded at various temperatures, as indicated. Peaks, corresponding to liquid-crystalline and gel lipids are indicated by * and **, respectively. (B) Chemical shift temperature dependencies recorded for narrow and broad peaks observed in 31P spectra of DPPC/CHAPS q* = 1.5 (blue and red), DMPC/CHAPS q* = 1.5 (green and brown), and POPC/CHAPS q* = 1.2 bicelles (yellow). Average linear approximation of temperature dependencies is plotted by the dashed lines.

The reduced size of bicelles with liquid-crystalline lipids, observed below the critical temperature, may be explained, assuming that smaller bicelles have the lower propensity to undergo phase transition than the bigger particles. Bicelles are not a continuous media; they represent a set of individual particles, which follow some kind of size distribution. In turn, rim detergents are rather mobile and may disturb the packing of lipids in IsoBs, which are in direct contact with the rim. Smaller bicelles contain the greater amounts of detergents per lipid molecule, which further disturbs the lipid packing. Using the simple ideal CHAPS-based bicelle model,12 one could estimate that 4.0 nm bicelles contain 72 lipid molecules, with 22 (31%) lipids being in direct contact with the rim surfactants. In turn, 5.5 nm bicelles contain 178 lipids, with 36 (20%) interacting to the rim, and this implies the less distorted lipid bilayer and higher phase transition temperature. Thus, the phase transition inside the one particular bicelle is a stochastic event, which has the probability that may depend on the size of the particle. Therefore, there should be a stochastic distribution of bicelles between the fluid and gel phases within the same sample. In addition, there may be a difference in the free energy of CHAPS in the rim of bicelles in gel and liquid-crystalline phases. If the presence of CHAPS in the rim of gel-phase bicelles was less favorable than in the rim of the liquidcrystalline IsoBs, this would redistribute the detergent between the two subsets of particles. Gel bicelles would contain less detergent per lipid and become larger, while liquid-crystalline particles would be formed with the lower q* and become smaller. This assumption is in agreement with the concentration of monomeric detergent in our systems. In bicelles that do not undergo the phase transition (DMPC/CHAPS with q =

1.0), the concentration of monomeric CHAPS in solution is almost constant (3.5 mM) and does not depend on the temperature.12 In DPPC/CHAPS q* = 1.35 bicelles, the concentration of monomeric CHAPS starts to increase below the critical temperature from initial 3.0 mM, until it reaches its maximum of 7−8 mM at 15 C, which is equal to the CMC of CHAPS in the absence of lipids (Figure S2). Thus, smaller bicelles would remain in the fluid phase at the given temperature. When temperature is decreased, these particles may become frozen, which results in the observed changes in the populations of bicelles with gel and liquid-crystalline lipids. This may be tested by measuring the size of bicelles in the gel and fluid phases for the mixture prepared with smaller q*. At q* = 1.5, the fraction of liquid-crystalline lipids is too low at 20 °C to perform the accurate measurements. For this reason, we prepared the q* = 1.35 DPPC/CHAPS bicelles and recorded the 31P diffusion spectra at 20 and 25 °C. The radii of both the gel phase and liquid-crystalline bicelles decreased to 5.53 ± 0.19 nm (p = 0.02) and 3.85 ± 0.04 nm (p = 0.009) at 25 °C in comparison to the q* = 1.5 mixture (Figure 3). Upon cooling the system to 20 °C, the radius of gel phase bicelles had increased to 5.98 ± 0.47 nm; however, the low accuracy of data does not allow a definitive conclusion (p = 0.063), whereas the size of liquid-crystalline bicelles had clearly decreased to 3.53 ± 0.13 nm (p = 0.0012). Thus, these data are in agreement with the above-stated hypothesis. We need to point out here that the coexistence of IsoBs with gel and liquid-crystalline lipids has important practical consequences. First, IsoBs should not be applied for the structural studies below the critical temperature of the bilayer lipid. One would always prefer to have the homogeneous 3430

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Figure 5. Effect of the rim-forming detergent. (A) 31P NMR spectra of DMPC/DHPC q* = 0.75 (left panel) and DMPC/Facade-EPC q* = 1.5 (right panel) bicelles recorded at various temperatures, as indicated. Peaks corresponding to liquid-crystalline and gel lipids are indicated by * and **, respectively. Peak from DHPC is indicated by D, peaks from Facade-EPC phosphate moieties are indicated by P1 and P2. (B) Chemical shift temperature dependencies recorded for narrow and broad peaks observed in 31P spectra of DMPC/Facade-EPC q* = 1.5 (red and blue), DMPC/ CHAPS q* = 1.5 (yellow and green), DMPC/FACADE-EM q* = 1.5 (brown and cyan), and DMPC/DHPC q* = 1 bicelles (dark-green, phase transition in these bicelles is not fractional). Additionally plotted are the chemical shifts of Facade-EPC group P1 in DMPC/Facade-EPC q* = 1.5 bicelles (light green). Average linear approximation of temperature dependencies is plotted by the dashed lines. (C) The fraction of gel phase DMPC in bicelles (relative intensity of the broad peak in 31P spectra) is plotted for 283 (blue), 288 (red), and 293 (green) K for four kinds of bicelles with the same radius of 3.4 nm (below the phase transition temperature).

is reduced at low q* (Figure 2B). If we define the effective critical temperature (Te) as a point with 50% of bicelles in the gel state, then Te is reduced from 32.5 °C at q* = 1.5 to 17 °C at q* = 0.6. This is in agreement with our hypothesis, suggested above, that small bicelles are less likely to be “frozen” than the larger ones. The presence of phase transition even in smallest particles seems to in part resolve the long-lasting argument about the relevance of small IsoBs.5,24 As we show here, small bicelles are able to reproduce one of the major properties of the lipid bilayer membranes: gel to liquid-crystalline phase transition. Effect of Lipid Chain Length and Saturation. So far, we used the 31P NMR to study only the single bicelle system, namely, DPPC/CHAPS. However, the applicability of the approach and general character of the conclusions needs to be tested with the other kinds of lipids and rim-forming surfactants. For that reason, we investigated the effect of lipid fatty acids on the 31P spectra and phase transitions and prepared the DMPC/CHAPS and POPC/CHAPS mixtures. POPC/CHAPS q* = 1.2 bicelles behaved as was expected: only the single and narrow peak was observed in the 31P NMR spectra with the temperature coefficient of 3−3.5 ppb/K and chemical shifts close to the observed for the liquid-crystalline DPPC/CHAPS bicelles (Figures 4 and S3). In other words, no phase transition is observed for the POPC/CHAPS IsoBs, which is expected, because the phase transition temperature of POPC bilayers is below 0 °C. DMPC behaved similarly to DPPC in CHAPS-based bicelles: two 31P signals, broad and

samples, while IsoBs below the critical temperature would provide highly polydisperse solutions, with highly varied lipid packing properties. In addition, IsoBs cannot be utilized to investigate the effects of lipid phase transition on the properties of membrane proteins, for the same reasons. To summarize the paragraph, we show that the phase transition of bilayer lipids is fractional in IsoBs, small liquidcrystalline particles and large gel bicelles coexist in solution within the broad range of temperatures. Such a behavior is likely to be caused by the different propensity of small and large bicelles to undergo the phase transitions of bilayer lipids. Effect of q*. The effect of bicelle size (q* ratio) on the parameters of lipid phase transitions can be investigated in more detail to understand the properties of the smallest particles. For this purpose, we prepared several DPPC/CHAPS mixtures with q* varied from 0.6 to 1.5 and recorded the 31P spectra at temperatures ranging from 5 to 70 °C. As was revealed by this experiment, the fractional phase transitions do occur even in very small bicelles (e.g., at q = 0.6), when the radius of particles is equal to 2.7 nm (this is equivalent to q* = 0.3 DMPC/DHPC particles, which are widely applied for solution NMR spectroscopy of membrane proteins34). The chemical shift dependencies on the temperature of both gel and fluid phase bicelles are almost identical at all tested q* (Figure 2A). This indicates that no substantial difference is observed between the lipid packing parameters in small and large IsoBs. However, some properties of phase transition are different in smaller particles. In particular, the fraction of gel-phase bicelles 3431

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Figure 6. Lipid−protein nanodiscs. (A) Superposition of 31P NMR spectra recorded at 283, 293, and 303 K for DMPC/MSPD1 LPNs (one signal) and DMPC/Facade-EPC q* = 3.0 bicelles (three signals). (B) Chemical shift temperature dependencies recorded for peaks observed in 31P spectra of DMPC/Facade-EPC q* = 3.0 (blue ■), DMPC/FACADE-EM q* = 3 (red ◆), DMPC/DHPC q* = 1 bicelles (yellow ▼), and DMPC/MSPD1 LPNs (green ▲). The linear approximation of data for LPNs below and above the critical temperature of DMPC is shown by dashed and dotted lines.

narrow, appeared in NMR spectra below 20 °C at q* = 1.5 (critical temperature of DMPC bilayers equals 24 °C). The broad peak is shifted upfield and is characterized by the big temperature coefficient, 8 ppb/K. Like for DPPC, the efficient phase transition temperature is reduced for lower q* (17 °C at q* =1.5 and 8 °C at q*=1). Thus, we can conclude here that the behavior of 31P signals in NMR spectra is similar for all phosphatidylcholine lipids in bicelles and clearly reflects the phase transitions. Moreover, the relationships between the critical temperatures of the phase transition for various lipids in bilayers are reproduced in the IsoBs. Effect of the Rim-Forming Agent. Effect of the rimforming agents on lipid phase transition in isotropic bicelles is also very important. In our previous works, we characterized the bicelles formed by CHAPS, CHAPSO, DHPC, bile salt, Facade-EM, and Facade-EPC in mixtures with the DMPC phospholipid.12,20 The listed surfactants allowed obtaining the bicelles with various magnitudes of the temperature-induced growth and various ability to maintain the native fold of membrane protein water-soluble domains.20 Here we selected the most prospective detergents: DHPC, Facade-EM, and Facade-EPC and applied the 31P NMR spectroscopy to estimate the phase behavior and lipid/detergent mixing in IsoBs. In all three systems, the fractional phase transition was observed, with parameters of the 31P spectra similar to those of DMPC/CHAPS (Figure 5, panels A and B). The efficient critical temperature was in all cases depending on q* in a manner, described previously: the Te value was reduced at low lipid/detergent ratios (Figure S4). Like it was in the case of DPPC/CHAPS, for all tested detergents, phase transitions were observed even in the mixtures with the relatively small particles, applicable for solution NMR studies: q* = 0.45 DMPC/DHPC (Figure S5, 3.0 nm), q* = 1.0 DMPC/CHAPS (3.1 nm), and q* = 1.1 DMPC/Facade-EPC (2.9 nm). Thus, we can state that

lipid phase behavior is similar in bicelles, formed at various q* and by various surfactants. However, there was the major difference between all rimforming detergents observed with respect to the number of lipids in the fluid and gel phases at various temperatures. If we take bicelles of similar hydrodynamic radii (3.4 nm) at 10 °C, we find that in the DMPC/Facade-EPC bicelles the fraction of lipid in the gel state is maximal (95%), in the DMPC/DHPC mixture the amount of “frozen” bicelles equals 69%, while the DMPC/CHAPS and DMPC/Facade-EM provide only 45− 48% of bicelles in the gel phase (Figure 5C). It is noteworthy that the detergents that provide the least expressed temperature-induced growth of IsoBs (Facade-EPC and DHPC) retain the most expressed phase transition of bilayer lipids. This completely disproves our previous hypotheses that the temperature-induced growth of bicelles directly reflects the lipid phase transition and that Facade-EPC is mixed with the lipid in both the bilayer and rim areas of bicelles.12,20 Another important observation refers to the properties of the bicelle rim. DHPC and Facade-EPC contain the phosphatidylcholine moieties and are revealed in the 31P NMR spectra. In accordance with the NMR data, the behavior of rim signals in NMR spectra is completely different to the behavior of lipids. No phase transition is observed for the rim of IsoBs. Single and narrow peaks of DHPC and Facade-EPC are present in 31P spectra, their temperature-dependent behavior is similar to the behavior of liquid-crystalline DMPC phosphate groups. In other words, while the lipids are in the gel phase, the rim is always “liquid” (Figure 5B). This definitely implies the absence of the detectable mixing between the detergents and lipids in the bilayer area of bicelles, at least in the gel state. Such clear segregation and distinct temperature-dependent behavior of the detergent and lipid signals also implies the absence of lipid molecules in the rim of the bicelles. Rim and planar areas definitely have different lipid packing properties; therefore, a 3432

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Figure 7. Concentration affects the phase transition and temperature-induced growth of isotropic bicelles. (A) Chemical shift temperature dependencies recorded for wide and narrow peaks observed in 31P spectra of DPPC/CHAPS q* = 1.5 bicelles prepared at concentrations of 4% (red and blue), 1.3% (green and yellow), and 0.3% w/w (cyan and brown). Lines represent the result of third-order polynomial approximation of the experimental data. (B) The fraction of gel phase DPPC in q* = 1.5 DPPC/CHAPS bicelles, prepared at 4% and 0.3% w/w. (C) The radius of DMPC/CHAPS q* = 1.5 bicelles is plotted as a function of DMPC concentration at 40 °C, errors do not exceed the size of points on the plot. (D) Temperature dependence of the radius R of DMPC/CHAPS q* = 1.5 bicelles measured at 29.5 mM DMPC and at 5.8 mM DMPC, errors do not exceed the size of points on the plot. The behavior of bicelles at 5.8 mM of DMPC below the phase transition is related to the variation of the free CHAPS concentration with temperature.

NMR spectra of very large IsoBs and LPNs below the phase transition temperature corresponded to the position of the broad peak in NMR spectra of small IsoBs. Thus, we can conclude, that from the viewpoint of headgroups, the lipid packing properties are similar in the LPNs and large IsoBs. Additionally, we state that the phase transition of lipids may be observed via the 31P NMR spectroscopy even for such large particles with the radii close to or exceeding 5 nm. Considering LPNs as a reference bilayer-containing membrane mimetic, we can state that behavior of lipids in LPNs is also the same as in small IsoBs, according to the chemical shifts of signals in 31P NMR spectra. Parameters of Phase Transitions and TemperatureInduced Growth Depend on the Concentration of IsoBs. Apart from the temperature, lipid contents, and detergent, the overall lipid concentration is also known to be important for the phase behavior of both anisotropic and isotropic bicelles.38 To investigate the influence of this parameter on the phase behavior of IsoBs, we diluted the initial sample of DPPC/ CHAPS q* = 1.5 bicelles 10-fold (to 0.3% w/w from initial 4%) with the solution containing 3 mM of CHAPS, corresponding to the concentration of free CHAPS in such a mixture.12 This procedure did not affect the average size of bicelles at 35 °C, according to the 1H diffusion measurements and did not alter

separate peak should be observed for such rim-placed lipids, which is absent from the NMR spectra. 31 P NMR Spectra of Lipid−Protein Nanodiscs and Large IsoBs. The belt protein of lipid−protein nanodiscs (LPNs) may be also considered as the rim of a bicellelike particle.35 Taking into account that the phase transition was found to take place in DMPC LPNs,36 it would be interesting to compare the properties of 31P NMR spectra of these particles to IsoBs at various temperatures. There was no sign of fractional phase transition found, only the single peak was observed in NMR spectra at all temperatures in the range of 5− 60 °C. However, the temperature coefficients of 31P chemical shifts clearly reflect the presence of phase transition. While above the Tm, the signal follows the behavior of DMPC in liquid-crystalline DMPC/CHAPS or DMPC/Facade-EPC bicelles, below the critical point, the slope of the temperature dependence of chemical shift is increased and the properties of the 31P spectra are similar to those of the “frozen” bicelles (Figure 6B). Moreover, the temperature dependence of 31P chemical shifts in LPNs is almost identical to the dependence observed for the q* = 3 DMPC/Facade-EPC bicelles that have the same radius of ca. 4.9 nm20,36,37 and is to a high extent similar to the dependencies recorded for the q* = 3 DMPC/ Facade-EM and q = 1 DMPC/DHPC mixtures. Moreover, 31P 3433

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Thinking of the phenomenon that could explain all listed facts, we can suggest only the single hypothesis: IsoBs can merge upon collisions. Such fusion of bicelles should be reversible to explain that at any given temperature the equilibrium size of bicelles is observed. Thus, the process that results in the temperature-induced growth may be considered as a reaction of bicelle “oligomerization”. Higher concentrations favor the fused state, which explains the concentration dependence, and, at any given composition, the propensity of fusion is governed by the thermodynamics of the system, the free energy difference between the “fused” and “monomeric” states. If the free energy of rim allows the long existence of such fused bicelles then growth occurs. This explains the absence of growth in small bicelles; the free energy pit is too narrow for small bicelles, while in large bicelles the perforations and other rim irregularities may be formed to stabilize the merged state of IsoBs. The reason for the enhanced temperature dependence of the fusion is not clear and should be somehow related to the entropy gain or heat capacity changes of the system upon fusion. The propensity of fusion should also depend on the elasticity of collisions and, therefore, the packing of lipids in bicelles. This explains the absence of growth in the gel-phase bicelles and the effect of POPC. In addition, both the elasticity of collision and the parameters of free-energy profile of bicelles may depend on the type of particular rim-forming agent. Facade-EPC and DHPC that possess charged zwitterionic headgroups may be less prone to the bicelle fusion, while uncharged CHAPS and Facade-EM are more likely to interact across the rims of two colliding particles. The proposed hypothesis may also explain several facts regarding the behavior of large anisotropic bicelles. First of all, the spontaneous orientation of bicelles is known to occur only at temperatures higher than phase transition temperature of bilayer lipid, and reproducible orientation is obtained only at relatively high concentrations of bicelles (25% w/w).32 Apparently, below the critical temperatures and at low concentrations anisotropic bicelles are too small to orient spontaneously, while the fusion of bicelles at high temperatures and in concentrated samples results in the formation of very large particles that undergo the orientation at strong magnetic fields. Another fact is the ability of Facade-EPC detergent to support the bicelle formation at extremely high q*, while the other rim-forming surfactants at high q* fail to form bicelles and the other phase is observed.19 As we show here, Facade-EPC is resistant to bicelle fusion upon collision and supports the least distorted packing of lipids in the bilayer area, which explains the phenomenon. Thus, we state that the present work and suggested hypothesis allow one to explain the phenomenon of temperature-induced growth of IsoBs with no need for bringing into consideration the altered bicelle morphology or mixing between the lipids and detergents. The problem of mixing between the detergent and lipid in the “bilayer” area of bicelles is also crucial for the structural biology. One would like to mimic the properties of cell membranes, and if the detergents are present in the bilayer in high amounts, they can distort its structure. This would decrease the extent of identity between the bicelles and bilayer membranes and challenge the relevance of the experimental data, obtained in such an environment. To get the complete understanding of the bicelle structure, one needs to summarize the whole set of data regarding the IsoBs. To proceed, we need to define what is implied by the “substantial extent of mixing”. Such mixing is quite difficult to

substantially the efficient phase transition temperature of lipids (Figure 7, panels A and B). On the other hand, the dilution resulted in a much narrower phase transition: the fraction of bicelles in the fluid phase is decreased to zero already at 27 °C at 0.3% instead of 10 °C for 4% solution. Dilution as well affected the chemical shifts of 31P signals in NMR spectra. The chemical shift decay with the decrease of temperature becomes much steeper for the gel lipids at high dilution and appears to reach a plateau at −1.15 ppm and 20 °C. Altogether this may be interpreted by assuming that the parameters of phase transitions depend on the particle collision rate in solution, which explains the majority of effects, described above. Collisions may cause the melting of bicelles, which explains the presence of the fraction of bicelles in the liquid-crystalline state below the critical temperature. Additionally, collisions do not allow the complete freezing of lipids in the gel bicelles, which explains the less steep chemical shift dependence on temperature at high concentrations and the absence of plateau at low temperatures. This finding may also explain the previously reported extremely broad phase transitions measured for the q = 0.75 and q = 0.5 DMPC/DHPC bicelles by the infrared spectroscopy.24 These measurements were made at 400 mM concentration, which corresponds to ca. 20% w/w fraction and substantially exceeds all concentrations tested in the present work. The concentration also appeared to affect the temperatureinduced growth of IsoBs, which is observed above the critical temperatures of lipids in some kinds of bicelles.12,22 Gradual dilution of initial 10% w/w DMPC/CHAPS q* = 1.5 bicelle solution with 2.5 mM CHAPS revealed the decrease in the magnitude of the specified effect. The radius of bicelles at 40 °C decreased from 8 nm at 60 mM of DMPC to 4.34 nm at 1.8 mM, while below the phase transition (15 °C) it remained almost unchanged and equal to 3.7 nm (Figure 7, panels C and D). Thus, the 32-fold dilution allowed for the weakening of growth upon heating to 40 °C from initial 116% to 19%. This implies that the temperature-induced growth is also depending on the collision frequency of bicelles in solution. The consequences of this conclusion and the nature of the growth are discussed below.

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SUMMARY AND CONCLUSIONS To understand the nature and reasons of the temperatureinduced growth of IsoBs, one needs to summarize all findings, reported in the present work and in the literature. Now we know the following facts: (1) the growth of IsoBs upon heating is not observed in particles with radii less than 3.0 nm.12,20,22 (2) The effect depends on the lipid composition of bicelles: it is at most expressed for the unsaturated lipids (POPC).12 (3) The magnitude of the effect depends on the rim-forming detergent; maximal growth is observed for the CHAPS and Facade-EM, and minimal growth is observed for the Facade-EPC and DHPC. Detergents that provide the minimal temperatureinduced growth reveal the more pronounced phase transition of lipids in bicelles. Thus, growth is not related directly to the lipid phase transition. (4) The effect depends on the concentration of bicelles; at high concentration, bicelles grow much more rapidly with heat. (5) The growth of IsoBs is not likely to be the result of the mixing between the lipid and detergent: 31P spectra of bicelles that experience the growth and of bicelles that do not grow upon heating are almost identical, and the absence of growth at low concentrations of bicelles does not agree with the mixing hypothesis. 3434

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First of all, we show here that detergents always remain “liquid”, regardless of the state of lipids in bicelles. When two types of particles are observed, there is no additional peak of detergent found, which would correspond to the detergent molecules, “trapped” inside the gel bilayer, due to the mixing. The intensity ratio between the detergent and lipid peaks is always retained. Therefore, lipids and detergents are not mixed substantially in the gel phase IsoBs. Moreover, we observe that 31 P chemical shifts behave almost identically in large and small bicelles of the same composition above the critical temperature. In accordance with our data, 31P chemical shift difference is much greater between the bicelles formed by various rim detergents than between the small and large liquid-crystalline bicelles of the same composition (Figure S6, panels A and B). This suggests that if lipid/detergent mixing is present, it is similar in large and small bicelle particles of the same content. In addition, we studied the behavior of other lipid/detergent mixtures e.g., DMPC/SDS and found no sign of phase transition even for very large particles (5.5 nm). Moreover, the size of such mixed micelles does not fit to the ideal bicelle model with reasonable parameters; the rim thickness appeared to be equal to 3.1 ± 0.1 nm (Figure S6C), which exceeds even the length of the DMPC molecule (2.0−2.2 nm). For comparison, the thickness of CHAPS rim is as small as 1.1 nm.12 This experiment may be considered as a negative control, demonstrating that when an indeed substantial lipid/detergent mixing is present in bicelles or mixed micelles, it could be detected following the size of particles and 31P chemical shifts of the phospholipid. The main idea that we would like to contradict in the current work is that small IsoBs that are used in solution NMR studies are somehow different from LPNs and large bicelles. We think that our data demonstrates clearly that properties of large and small bicelles are similar. We do not observe any abrupt change in the behavior of low-q and high-q mixtures, which would allow one to postulate the distinct morphology of small and large particles. All bicelle parameters change gradually and do not contradict the “ideal bicelle model”. If lipid-detergent mixing is present, it is similar in very large and the smallest particles and permits one to preserve the patch of lipid bilayer that undergoes the phase transition.

detect, and it is also hardly possible to put forward any kind of quantitative cutoff for the majority of bicelle systems. Therefore, we propose that the presence of substantial mixing can be postulated if the behavior of bicelles fails to reproduce any aspect of the behavior of lipid bilayers or particles that were shown to contain the patch of the lipid bilayer, such as the LPNs. These aspects may include the lipid packing properties, lipid phase transitions, and behavior of membrane proteins inside the bicelles. The presence of lipid bilayer in the large IsoBs (q > 0.75 for DMPC/DHPC) was shown by a variety of approaches. Direct observation of particles with cryoelectron microscopy revealed the discoidal shape of the bicelles in solution,5,38 large bicelles were shown to undergo the gel to liquid-crystalline phase transitions at temperatures, close to the observed for bilayer lipids,24 and, finally, most membrane proteins are able to sustain the native fold and activity in the bicelle environment.39,40 Thus, the main question that needs to be answered is do the small bicelles used in structural studies behave in the same way as the large bicelles and LPNs? Until recently, there were two major effects that suggested the mixing of lipids and detergents in IsoBs. First, several studies stated that the size of small bicelles does not fit the socalled “ideal bicelle model”.5,39 However, the refined models that take into account the shape of the bicelle rim12,23 revealed that size of small bicelles perfectly follow the theoretical predictions. Actually, the large IsoBs demonstrated the “deviant” behavior, the so-called temperature-induced growth.12 However, according to the current work, this effect has a pronounced concentration dependence. Therefore, we suggested that the growth is a result of the reversible fusion of bicelles. Second, the phase transition of lipids was thought to be absent in small IsoBs.24 The phase transition is a fundamental property of lipid bilayer, therefore the absence of such transition was considered as the evidence in favor of the absence of the bilayer itself in IsoBs. This problem seems to be also resolved in the current work: we showed that the phase transitions of lipids are observed even in the smallest bicelles, which are applied in the structural studies. On the other hand, the properties of the phase transitions in small IsoBs are peculiar: these transitions are “fractional” and reveal the reduced critical temperature, which depends on the size of the particles. When gel and fluid phase particles coexist in solution, the size of gel phase bicelles is much greater than the size of the liquid-crystalline particles, which stands against the lipid packing properties in gel and liquid-crystalline bilayers. These phenomena may be caused by the presence of lipid/ detergent mixing; however, as we show above, another explanation is more likely to be relevant. The mixing hypothesis fails to account for some of the reported data: the concentration dependence of the phase transition properties in small IsoBs and the greater size of liquid-crystalline IsoBs, when the whole population of bicelles is considered above the critical temperature. Above, we provide several possible explanations of the phase transition properties in small IsoBs, based on the broad bicelle size distribution, destabilizing effects of the rim detergent, and bicelle collisions. Therefore, we can state that there is no data up to date, that does not agree with the presence of lipid bilayer in the planar area of small IsoBs. This certainly does not mean that there is at all no mixing between the lipids and detergents in IsoBs. However, there is some indirect data that suggests that if such mixing is present, it is not strong enough to alter the lipid packing properties in IsoBs, in comparison to large bicelles, LPNs, and lipid bilayers.

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ASSOCIATED CONTENT

S Supporting Information *

Supporting Information includes six additional figures. This material is available free of charge via the Internet at http:// pubs.acs.org/. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ acs.langmuir.7b03610. Results of the deconvoluton of 31P NMR spectrum of DPPC/CHAPS q* = 1.5 bicelles at 298 K; concentraton of CHAPS in the monomeric form is ploted as a functon of temperature for q* = 1.35 DPPC/CHAPS (blue), and q* = 1.0 DMPC/CHAPS (red) bicelles, 31P NMR spectra of DMPC/CHAPS q* = 1.5 bicelles at 288 and 293 K are shown together with the results of their deconvoluton by two Lorentz lines; fraction of gel phase DMPC/DHPC bicelles (relatve intensity of broad peak in 31P spectra) is plotted as a functon of temperature for q* = 1 (green ▲), 0.75 (yellow ▼), 0.6 (red ◆), and 0.45 (blue ■); and 1P NMR spectra of DMPC/DHPC bicelles prepared with q* = 0.2, 0.45, 0.6, 0.75, and 1.0 3435

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recorded at 278 (red), 283 (blue), and 288 (green) K, and 31P chemical shifts depend on the parameters of lipid packing in bicelles (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Konstantin S. Mineev: 0000-0002-2418-9421 Author Contributions

The manuscript was written through the contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The work is supported by the Russian Science Foundation, Grant 14-14-00573. Experiments were partially carried out using the equipment provided by the IBCH core facility (CKP IBCH, supported by Russian Ministry of Education and Science, Grant RFMEFI62117X0018).

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ABBREVIATIONS NMR, nuclear magnetic resonance; LPN, lipid−protein nanodisc; IsoB, isotropic bicelle; DMPC, 1,2-dimyristoyl-sn-glycero3-phosphocholine; DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; DHPC, 1,2-dihexanoyl-sn-glycero-3-phosphocholine; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; SDS, sodium dodecyl sulfate

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REFERENCES

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