Immersion Depths of Lipid Carbons in Bicelles ... - ACS Publications

Article pubs.acs.org/JPCB

Immersion Depths of Lipid Carbons in Bicelles Measured by Paramagnetic Relaxation Enhancement Jobst Liebau* and Lena Mal̈ er Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden S Supporting Information *

ABSTRACT: Myriads of biological processes occur in or at cellular lipid membranes. Knowledge about the localization of proteins, lipids, and other molecules within biological membranes is thus crucial for the understanding of such processes. Here, we present a method to determine the immersion depths of lipid carbon atoms in membranes by paramagnetic relaxation enhancement (PRE) caused by the presence of doxylated lipids. As membrane mimetics, we employ small isotropic bicelles made of synthetic lipids and of natural Escherichia coli phospholipid extract. Bicelles are particularly suitable for solution state NMR since they maintain a lipid bilayer while they are at the same time amenable to solution state NMR experiments. PREs were measured in the presence of different doxylated lipids with the nitroxide radical located in the headgroup and at various positions in the acyl chain. Theoretical PREs were calculated assuming a simple bicelle model using the Solomon−Bloembergen equations. Immersion depths of the lipid carbon atoms were obtained by a least-squares fit of the theoretical to the experimental PREs. The carbon immersion depths correspond well to results obtained by other methods and differences do not exceed 3−5 Å. This means that the method presented here provides sufficient resolution to distinguish the localization of carbons in different regions of the lipid bilayer, despite considerable simplifications of the underlying theory. These simplifications include a simple form of the spectral density function, which we find is sufficient to reliably determine immersion depths. A more complicated spectral density function that includes bicelle, lipid, and local motions may only improve the results if its parametrization is good enough. The approach presented here may be extended to the determination of protein localization in membranes employing realistic membrane mimetics like the bicelles made of E. coli phospholipid extract used here.

■

INTRODUCTION

also nitroxide spin-labels linked to lipids at different positions in the acyl chain or the headgroup have been used.21,24,30 For most detailed studies of membrane properties, simplified lipid systems are required. Small isotropic bicelles are mixtures of lipids and detergents, in which the lipids form a central diskshaped bilayer which is solubilized in aqueous solvent by detergents located in the rim of the bicelle.31−33 This idealized picture of a small isotropic bicelle is approached for lipiddetergent ratios, called q-values, of 0.5 to 1. For higher q-values, large bilayer structures are formed that align in magnetic fields.34−37 For q-values below 0.5 the spatial separation between lipids and detergents is most likely lost.38,39 Small isotropic bicelles are particularly suitable as membrane mimetics for solution state NMR, since they, in contrast to detergent micelles, contain a membrane bilayer. The strong curvature of micelles can lead to structural distortions of proteins and may impair protein function.33,38 In contrast, bicelles have been shown to maintain native-like structure and function of proteins.40−43 At the same time, isotropic bicelles are sufficiently small to be suitable for

Biological membranes form a protective barrier against the environment but have functional roles as well1,2 and about onethird of all proteins are believed to be membrane-associated.3 As a consequence, a multitude of essential biological processes occur in or at membranes. Membrane immersion depths of molecules are thus an important aspect for the understanding of membrane related cellular processes. For instance, proper membrane insertion4 and adequate membrane environments5 are required for the function of membrane proteins. Moreover, the degree and localization of unsaturations as well as the length of the lipid acyl chains determine the fluidity of membranes.6 Membrane immersion depth measurements can be carried out using a multitude of methods, like the fluorescence based parallax method,7,8 small-angle neutron or X-ray scattering methods (SANS/SAXS),9−13 electron paramagnetic resonance,14−17 and NMR.18−24 Long distances or immersion depths can be measured by NMR using paramagnetic relaxation enhancement (PRE), which occurs when a paramagnetic molecule comes into proximity (5−35 Å) of the observed nucleus enhancing nuclear relaxation rates.25 Frequently, soluble paramagnetic molecules like Mn2+,26 O2,27 or chelated lanthanides28 are employed,29 but © 2017 American Chemical Society

Received: June 14, 2017 Revised: July 12, 2017 Published: July 14, 2017 7660

DOI: 10.1021/acs.jpcb.7b05822 J. Phys. Chem. B 2017, 121, 7660−7670

Article

The Journal of Physical Chemistry B

temperature. Likewise, organic solvents of either TEMPO-, 5doxyl-, 10-doxyl-, or 16-doxyl PC were evaporated under a stream of N2-gas. 500 μL of bicelle solution was then added to each of the spin-labels and the solution was thoroughly vortexed. The final sample specifications were as follows: 50 mM lipids, 2 mM spin-label, and 100 mM DHPC-d22 in 50 mM sodium phosphate buffer at pD 7.4 in 100% D2O. Three bicelle types with different lipid composition were studied: (1) zwitterionic bicelles with 100% DMPC, (2) anionic bicelles with 70% DMPC and 30% DMPG, and (3) E. coli bicelles containing 100% E. coli phospholipid extract. 31 P and 13C Experiments. 31P and 13C spectra were acquired on a 600 MHz Bruker Avance spectrometer (13C frequency 151 MHz, 31P frequency 243 MHz) equipped with a triple resonance probe-head. Relaxation Experiments for PRE Measurements. Natural abundance 13C longitudinal relaxation rates were measured on a 600 MHz Bruker Avance spectrometer (13C frequency 151 MHz) using a 1D 13C-detected inversion recovery pulse sequence for zwitterionic and E. coli bicelles. Ten delays ranging between 0.005 and 6 s were acquired with at least 2000 scans per experiment. For anionic bicelles, 8 2D 13C/1H correlation spectra were acquired with increasing 13C T1 relaxation delays ranging between 0.005 and 1.4 s. 1H decoupling was used throughout the experiments. All experiments were carried out at 25 °C. Peak integrals were fitted to a monoexponential function to obtain the longitudinal relaxation rate R1. The experimental paramagnetic relaxation enhancements Γ1,exp were obtained by subtracting the longitudinal relaxation rates measured for a reference sample containing no spin-label (R1,ref) from the longitudinal relaxation rates of samples containing spin-label (R1,label), i.e.

solution state NMR applications. Historically, the lipid component of bicelles was often 1,2-dimyristoyl-sn-glycero-3phosphocholine (DMPC) but bicelles with varying lipid compositions can be produced to more faithfully mimic different types of natural membranes. For example, bicelles can be enriched in anionic lipids,44−46 like dimyristoyl-sn-glycero-3phosphoglycerol (DMPG), galactolipids can be introduced,47 and the degree of unsaturation5 and the acyl chain length48 can be varied. To mimic natural membranes, we recently characterized bicelles that consisted of natural phospholipid extract from Escherichia coli.49 These novel bicelle systems are realistic mimics of the phospholipid composition of E. coli inner membranes that consist of three headgroup types: the zwitterionic phosphatidylethanolamine (PE), which is most abundant (about 70−80% of the lipids), and the anionic phospholipids phosphatidylglycerol (PG, about 10−20%) and cardiolipin (about 5%).2,50,51 The acyl chains show substantial variations in length and degree of unsaturation. In the strain used in this study, 16:0 acyl chains are most common and about 30% of the acyl chains contain unsaturations or cyclopropanations under standard growth conditions.52,53 Here, we present a solution state NMR method to measure the immersion depths of lipid carbon atoms in bicelles made from synthetic lipids and from E. coli phospholipid extract. We employ lipids with nitroxide spin-labels in the headgroup and at various positions in the acyl chain to measure PREs of the carbons’ longitudinal relaxation rates R1. From such measurements, quantitative estimates of the immersion depths of the carbon atoms in the bilayer are obtained. The results can serve as an “immersion depth ruler” in membranes. They will thus be useful for studies of the immersion depth of proteins and peptides to which the approach presented here may be extended.

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Γ1,exp = R1, label − R1, ref

EXPERIMENTAL SECTION Materials. Tail-doxylated lipids (1-palmitoyl-2-stearoyl-(5-/ 10-/16-doxyl)-sn-glycero-3-phosphocholine, abbreviated as 5-/ 10-/16-doxyl PC), 1,2-dipalmitoyl-sn-glycero-3-phospho(tempo)choline (TEMPO-PC), tail-deuterated 1,2-dihexanoylsn-glycero-3-phosphocholine (DHPC-d22), 1,2-dimyristoyl-snglycero-3-phosphocholine (DMPC), and 1,2-dimyristoyl-snglycero-3-phosphoglycerol (DMPG) were purchased from Avanti Polar Lipids (Alabaster, AL). Purification of Phospholipids from AD93WT. Lipids were purified from E. coli by the Folch method54 as described earlier.49 Briefly, AD93WT strains55 were grown at 20 °C from an overnight culture for 24 h in 2x LB with 25 μg/mL kanamycin and 34 μg/mL chloroamphenicol. Cells were then harvested and resuspended in 20 mM PIPES, 1 mM ethylenediaminetetraacetic acid, 150 mM NaCl, 0.0002% NaN3 buffer at pH 7.4. Lipids were extracted by washing in chloroform:methanol 2:1 (v/v) and were washed again in the aforementioned buffer to remove divalent ions. The chloroform phase was collected and membrane components were separated on a silica gel column. Diacylglycerol and fatty acids were eluted with chloroform and phospholipids were subsequently eluted with methanol. The phospholipids were kept at −20 °C in a 2:1 (v/v) chloroform:methanol solution until further use. Bicelle Preparation. Organic solvents of an appropriate amount of lipid stock solution were evaporated under a stream of N2-gas. 100 mM DHPC-d22 from a 1 M stock solution and 50 mM sodium phosphate buffer at pD 7.4 in 100% D2O were added and the solution was thoroughly vortexed. It was then subjected to three cycles of freezing at −20 °C and thawing at room

(1)

Paramagnetic Relaxation Enhancement. Paramagnetic relaxation enhancement (PRE) occurs when the observed nucleus is located in the proximity of an unpaired electron. The paramagnetic relaxation enhancement Γ1 of the longitudinal relaxation rate R1 is described by the Solomon−Bloembergen (SB) equations and is at reasonably high magnetic field given by56−59 2 ⎛ μ0 ⎞ 2 2 2 ⎜ ⎟ γ g μ S(S + 1)1(ω ) C 5 ⎝ 4π ⎠ C e B 2

Γ1 =

(2)

where the r−6 distance dependence of the PRE between nucleus and unpaired electron is included in the spectral density function 1 (ωC) and the other constants are μ0, the magnetic permeability of vacuum; γC, the gyromagnetic ratio of carbon; g, the electron gfactor; μB, the electron Bohr magneton; and S, the electron spin quantum number. The spectral density function depends on the dynamics of the system under investigation that mediate relaxation. In a first approximation, we assume that the dynamics can sufficiently be described by a single correlation time τr: τc 1SB(ωC ) = ⟨r −6⟩ and τc 1 + (ωC τc)2 = (τr −1 + τM −1 + τS−1)−1

(3)

Here ωC is the carbon frequency at the externally applied magnetic field, τM is the liftetime of the transient complex between lipid and spin-labeled lipid, and τS is the electron relaxation time, which is several hundreds of nanoseconds for nitroxide radicals.60,61 Since ωS ≫ ωC, the contribution of the 7661


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The Journal of Physical Chemistry B spectral density function at the electron frequency ωS can be neglected. Combining eq 3 and eq 2 yields

Γ1, calc = C⟨r −6⟩

Table 1. Immersion Depths of Spin-Labels and Their Distribution in 1-Palmitoyl-2-oleoyl-sn-glycero-3phosphocholine (POPC) Bilayers Computed by MD Simulations

(4)

where all constants have been collected in the constant C. C can be determined from knowledge of all the constants; however, in particular, the correlation time τc is not necessarily known a priori. Therefore, C is treated as a global variable in the fitting procedure. Equation 3 assumes that the dynamics that cause PREs are sufficiently described by a single correlation time. However, lipids in bicelles undergo complex dynamics. The model-free approach was originally developed to describe protein backbone62,63 or surfactant64−66 dynamics and has also been employed to incorporate the influence of local motions on PREs.25 Moreover, the model free approach has been extended to describe lipid dynamics in bicelles.15,67 Assuming that local, lipid, and bicelle dynamics occur on different time scales the extended model-free (MF) spectral density function is given by

+ 1SBMF ,3) = ⟨r ⟩1SBMF ,1 − 3

distribution of the immersion depth/Å

reference

TEMPO-PC 5-doxyl PC 10-doxyl PC 16-doxyl PC

14.4a 12.8 10.0 1.0b

4.4 2.9 3.4 4.2

68 68 68 11, 68, 70, 71

The immersion depth of TEMPO-PC is concentration dependent. For low concentrations, as in this study, the doxyl group is located closer to the bilayer center than for higher concentrations of the spinlabel; i.e., the TEMPO-PC headgroup bends back into the bilayer at low concentrations.68 bThe immersion depth was not simulated for 16-doxyl PC in ref 68. Instead, the position of the terminal carbon11,70,71 is assumed to be the doxyl position and the distribution is assumed to be the same as for 14-doxyl PC in ref 68.

other lipids were set by randomly generating initial immersion depth values restricted by generous upper and lower bounds. From this bicelle model Γ1,calc was computed using eq 4 by summing the contribution of all lipids in one leaflet to the total PRE considering the spin-labeled lipids in both leaflets. Having computed Γ1,calc, the energy function given by

(1 − Sloc 2)τloc ⎞ ⎟ = ⟨r −6⟩(1SBMF ,1 + 1SBMF ,2 1 + (ωC τloc)2 ⎟⎠ −6

distance from the bilayer center/Å

a

⎛ S 2S 2τ (Sloc 2 − Slip 2Sloc 2)τlip lip loc bic 1SBMF (ωC ) = ⟨r −6⟩⎜⎜ + 2 1 + (ωC τlip)2 ⎝ 1 + (ωC τbic) +

spin-label

N

E=

(5)

∑ doxyls

where Slip2 and Sloc2 are the squared order parameters of the lipid and the local 13C − 1H bond motion respectively, τlip and τloc are the correlation times of the lipid and local motion and τbic is the correlation time of the entire bicelle. 1 SBMF,1, 1 SBMF,2, and 1 SBMF,3 denote the three terms of the extended spectral density function. For rigid lipids and 13C − 1H bonds, Slip2 and Sloc2 are 1 and 1 SBMF(ωC) reduces to 1 SB(ωC). Note that in this case τbic is not necessarily identical to τc as discussed below. Order parameters and correlation times are obtained from measurements of relaxation rates, typically the longitudinal relaxation rate R1, nuclear Overhauser enhancements, and the transverse relaxation rate R2. Employing the spectral density function of the model free approach 1 SBMF(ωC) instead of 1 SB(ωC) in eq 2 thus provides a more comprehensive description of the lipid dynamics that may modulate the orientation of the 13C−e− vector and thus could give rise to dipole−dipole relaxation of the carbon nuclear spins caused by the presence of a paramagnetic molecule. However, application of 1 SBMF(ωC) does not allow a simplification of the type given by eq 4; i.e., prior knowledge of the order parameters and correlation times is required to apply eq 5 in combination with eq 2. Fitting Procedure. The fitting was done with an in-house Matlab program that minimizes the distance squared between the measured paramagnetic relaxation enhancements Γ1,exp and theoretical paramagnetic relaxation enhancements Γ1,calc. In order to compute Γ1,calc, the bicelle was modeled as a disk of 4.5 nm radius that contains 68 cylindrically shaped lipids in each leaflet. One lipid per leaflet was randomly chosen to contain the spin-label. The mean immersion depths of nitroxides in spinlabeled lipids as well as the immersion depth distribution used in this study is given in Table 1.68 The position of the spin-label was randomly set to a fixed immersion depth d from the bilayer center based on this information. The depths of the carbon atoms in the

k

∑ (∑ Γ1, calc(xi , yi , zN , C) − Γ1,exp)2 1

i≠j

(6)

was minimized by varying the immersion depths zN of the N carbon atoms (N local variables) and C (1 global variable). Here the sum is over all samples with different nitroxide spin-labels (doxyls), over all N observable carbon atoms and all k lipids in one leaflet of the bicelle except the spin-labeled one.82 Since the sum is over all lipids, the value of the energy function depends only on the immersion depths zN of the carbon atoms and on C. Γ1,exp was measured for 3 or 4 spin-labeled lipids, and therefore 3N (or 4N) data points were available to fit N + 1 parameters (N carbon immersion depths and C). It is not expected that a single fit provides a good estimate of the immersion depths since Γ1 depends on the mean distance ⟨r−6⟩ between the carbon atoms and nitroxide spin-labels of the ensemble. Therefore, the fitting procedure was repeated multiple times in order to cover the conformational space of spin-labels in the sample including their locations inside the bicelle and their immersion depths d. The calculated PREs and immersion depths were thus the mean values for all simulated bicelles. The improvement of the fit by increased sampling of the spin-label conformational space was monitored by the quality factor Q, which measures the rootmean-square distance between calculated and experimental data normalized by the experimental data. The quality factor (Qfactor) is defined as25,69 N

Q=

k

∑doxyls ∑1 (∑i ≠ j Γ1, calc(xi , yi , zN , C) −Γ1,exp)2 N

∑doxyls ∑1 Γ1,exp2

(7)

where the mean of the calculated paramagnetic relaxation enhancements is taken over all iterations, i.e., simulated bicelles. There is a direct relation between Pearson’s R-factor and the Qfactor, but the Q-factor is often preferred for highly correlated NMR-data.69 7662


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Figure 1. Nomenclature of (A) the headgroup carbons in phosphatidylcholine (PC), PE, and PG, (B) of the glycerol and acyl chain carbons and (C) of acyl chain modifications found in E. coli, which are I. unsaturations and II. cyclopropanations. R represents the lipid rest in (B) and R′ is an insertion into the acyl chain. Note that Cω3 = C12, Cω2 = C13, and CH3  C14 in DMPC and DMPG. In contrast, a broad range of acyl chain lengths is found in E. coli phospholipids.53

Figure 2. Paramagnetic relaxation enhancements of carbons in zwitterionic bicelles for samples doped with (A) TEMPO-PC, (B) 5-, (C) 10-, and (D) 16-doxyl PC. Experimental PREs (Γ1,exp) are in blue. Calculated PREs (Γ1,calc) are in red and were obtained using eq 4.

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RESULTS

immersion depths were obtained by minimization of eq 6. For nomenclature of the lipid carbons see Figure 1. The experimentally determined PREs for zwitterionic bicelles in the presence of the four spin-labeled lipids employed in this study are shown in Figure 2 (blue bars) and the experimental data for anionic and E. coli bicelles are provided in the Supporting Information, Figures S3 and S5, respectively. Qualitatively, these data give an impression of where the different observable carbons are located in the bilayer provided that the spin-label position is known (see Table 1). The carbons of the glycerol backbone and the initial carbons of the acyl chain are located close to the

We determined the immersion depths of lipid carbons in bicelles for three different systems with q-values of 0.5:100% DMPC/ DHPC-d22 (zwitterionic bicelles), 70% DMPC and 30% DMPG/ DHPC-d22 (anionic bicelles), and E. coli phospholipid extract/ DHPC-d22 (E. coli bicelles). 13C and 31P spectra with peak assignments are shown in the Supporting Information, Figures S1 and S2, respectively. PREs in bicelle systems containing TEMPO-PC, 5- and 10-doxyl PC and additionally 16-doxyl PC for zwitterionic and E. coli bicelles were measured experimentally and theoretical PREs were computed using eq 4. Carbon 7663


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The Journal of Physical Chemistry B paramagnetic centers of TEMPO-PC and 5-doxyl PC. On the other hand, the terminal carbons in the acyl chain are located closer to the 10- and 16-doxyl PC spin-labels. The general picture is the same for anionic and E. coli bicelles. Note that the measurement uncertainties are high. The reason for this is that Γ1,exp is the difference of two relaxation rates (see eq 1), while measurement uncertainties add up due to error propagation. Immersion depths were obtained by fitting computed PREs to the experimental data. To obtain meaningful values the conformational space of the spin-labels inside the bicelle bilayer needed to be sampled; i.e., the spin-label position in the bicelle and the immersion depths of the radical were randomly varied. The Q-factor, which describes the correlation between experimental and computed PREs, is shown in Figure 3 for

Table 2. Immersion Depths of Lipid Carbons in Zwitterionic, Anionic, and E. coli Bicellesa distance from the bilayer center/Å carbon atom

zwitterionic bicelles

anionic bicelles

E. coli bicelles

PC-γ PC-β PC-α PE-β PE-α PG-γ PG-β PG-α g3 g2 g1 C2 C3 C−C CC Δc Δt Cω3b Cω2b CH3

20 ± 4 19 ± 4 20 ± 4 n.a. n.a. n.a. n.a. n.a. 15 ± 3 18 ± 4 13 ± 3 12 ± 3 12 ± 3 n.a. n.a. n.a. n.a. 5±4 5±4 4±4

22 ± 4 20 ± 4 22 ± 4 n.a. n.a. 14 ± 2 14 ± 2 15 ± 3 15 ± 3 18 ± 3 17 ± 4 12 ± 3 14 ± 2 n.a. n.a. n.a. n.a. 3±5 3±4 3±4

21 ± 5 20 ± 4 20 ± 5 18 ± 4 19 ± 4 15 ± 5 18 ± 4 21 ± 4 21 ± 3 17 ± 4 13 ± 3 12 ± 3 11 ± 3 7±6 8±7 8±6 8±6 4±5 4±5 4±5

a

For nomenclature see Figure 1. bThe nomenclature refers to the carbons preceding the methyl group. For E. coli bicelles, the chains may or may not be saturated at C9 or C11. For zwitterionic and anionic bicelles they are saturated and Cω3 = C12, Cω2 = C13, and CH3C14. Figure 3. Q-factor for the PRE-analysis of carbon atoms in zwitterionic bicelles when the computed PREs are averaged over an increasing number of bicelles. The Q-factor is computed according to eq 7.

For an extended PRE-analysis of zwitterionic bicelles we used dynamical parameters (Slip2, τlip, Sloc2, τloc) from a similar bicelle system, where bicelles were doped with galactolipids.47 The previously unpublished dynamical parameters for DMPC in such bicelles were obtained as described in ref 47 and are shown in Table S1 in the Supporting Information. DMPC R1 values of the bicelles doped with galactolipids are similar to R1 values measured in the absence of spin-labels for zwitterionic bicelles in this study. Moreover, the dynamical parameters of DMPC carbons in galactolipid bicelles agree well with previously published results for purely zwitterionic bicelles.48 Therefore, we assume that the presence of galactolipids does not significantly influence the dynamics of DMPC lipids. On the basis of these results we obtained immersion depth estimates employing the spectral density function as given in eq 5. In Table 3 the immersion depths of lipid carbons in zwitterionic bicelles obtained using the simple form of the spectral density function eq 3 are compared to the results when the more comprehensive form of eq 5 is employed. Systematic differences were observed for the glycerol and the PC headgroup carbons and slight differences were observed for the acyl chain carbons. Experimental and computed PREs and the quality factor at increasing numbers of simulated bicelles are shown in the Supporting Information, Figures S7 and S8, respectively.

zwitterionic bicelles for increasing numbers of simulated bicelles. The Q-factor improved dramatically when the average PRE values for about 30 bicelles were considered. After simulation of about 150 bicelles the Q-factor stabilized indicating that the conformational space of different spin-label positions and immersion depths had been sufficiently sampled. Q-factors for the fit of carbon immersion depths of anionic and E. coli bicelles are found in the Supporting Information, Figures S4 and S6, respectively. The Q-factor for the PRE-analysis of zwitterionic bicelles was below 0.3 implying a strong correlation between theoretical and experimental data. The Q-factors for anionic bicelles and E. coli bicelles were similar. The results of the fittings are shown in Table 2. The PC headgroups were found to be located about 20 Å, and thus farthest away from the bicelle center. PE and PG headgroups in E. coli bicelles have similar immersion depths, while PG headgroups in anionic bicelles appeared to be somewhat closer to the membrane center (distance from the bilayer center ∼14 Å) than the other lipid headgroups. The results demonstrate that the glycerol backbone is roughly located at 16 Å from the bicelle center and the initial carbons of the acyl chain just below. Unsaturated carbons and cyclopropanations in E. coli lipids are located at an intermediate distance of 8 Å from the bicelle center and the terminal carbons are 3−5 Å away from it. The uncertainty in the determination of the position is high, especially for the acyl chain carbons. This distribution is not necessarily a measurement uncertainty but may relate to the fact that due to lipid dynamics carbon positions are not well-defined (see discussion).

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DISCUSSION Bicelle Characteristics. In this study, we measured the immersion depths of carbon atoms in small isotropic bicelles employing distance-dependent PREs caused by paramagnetically labeled lipids with which the bicelles were doped. Experiments were conducted on bicelles with a lipid-detergent ratio (q-value) of 0.5 at pD 7.4 and at 25 °C. The DHPC concentration was 100 7664


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The Journal of Physical Chemistry B Table 3. Distance from the Bilayer Center of Lipid Carbons in Zwitterionic Bicelles Computed Using 1 SB(ωC) and 1 SBMF(ωC)a

Table 4. Distances from the Bilayer Center of Carbons in Zwitterionic Bicelles Compared to Results from Other Methodsa distance from the bilayer center/Å

distance from the bilayer center/Å PC-γ PC-β PC-α g3 g2 g1 C2 C3 Cω3b Cω2b CH3b

1 SB method

1 SBMF method

20 ± 4 19 ± 4 20 ± 4 15 ± 3 18 ± 4 13 ± 3 12 ± 3 12 ± 3 5±4 5±4 4±4

23 ± 2 24 ± 2 25 ± 2 26 ± 2 28 ± 2 25 ± 2 14 ± 4 12 ± 3 4±4 4±4 2±4

zwitterionic bicelles (this study)

16:0/18:2 PCb,71

POPC11

carbon atom

NMR

MD

MD

SANS/SAXS

PC-γ PC-β PC-α g3 g2 g1 C2 C3 Cω3c Cω2c CH3c

20 ± 4 19 ± 4 20 ± 4 15 ± 3 18 ± 4 13 ± 3 12 ± 3 12 ± 3 5±4 5±4 4±4

19.5 ± 3.6 19.4 ± 2.9 19.5 ± 2.5 17.2 ± 2.0 n.a. n.a. n.a. n.a. 3.2 ± 1.7 2.1 ± 2.4 n.a.

22 n.a. n.a. 19 17 16 14 14 4 3 2

22 22 22 16 16 16 n.a. n.a. n.a. n.a. 0

a For nomenclature see Figure 1. bFor DMPC Cω3 = C12, Cω2 = C13, and CH3C14.

mM and thus exceeded the critical micelle concentration of DHPC, which is 15 mM,72 by almost 1 order of magnitude. The properties of small isotropic bicelles have been intensely characterized at varying temperatures, concentrations and compositions.33 It has been concluded that small isotropic bicelles persist for q-values between 0.5 and 1, when the detergent concentration is substantially higher than the detergent’s critical micelle concentration, in a temperature range from 10−40 °C, and at neutral pH.33 The bicelles employed here, are thus expected to be small enough to reorient isotropically in solution. Yet, the degree of mixing between lipids and detergents and thus the question of whether or not a lipid bilayer exists in small isotropic bicelles is a matter of debate.32,72,73 As shown in Figure S2, DMPC and DHPC in zwitterionic and anionic bicelles show distinct peaks in 31P spectra; i.e., the headgroups experience different chemical environments. Further experimental evidence from other studies suggests that the detergents localize in a micelle-like environment in the rim and that lipids form a bilayer in the center of the bicelle.32 Still, some mixing between detergents and lipids may occur.73 Previously, we characterized E. coli bicelles and found that their size is comparable to similar bicelle systems made of synthetic lipids. Moreover, lipid dynamics were similar to DMPC/DHPC bicelles.49 Therefore, we concluded that also E. coli bicelles can be characterized as small isotropic bicelles. Localization of Lipids in Bicelles. Lipid bilayers of different compositions have been intensely studied in silico by molecular dynamics (MD) simulations and by SANS and SAXS. PC lipids of different chain length11,74 and degree of unsaturation11,70,71 have been investigated. Also, studies of membranes enriched in negatively charged lipids75 or 1-palmitoyl-2-oleoyl-sn-glycero-3phosphoethanolamine (POPE)74,76 have been conducted. Tables 4 and 5 compare the carbon immersion depths found here with immersion depths from representative MD studies,71,76,77 and with data from SANS/SAXS studies of carbon immersion depths in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),11 DMPG,75 and 1-palmitoyl-2-oleoyl-snglycero-3-phosphoglycerol (POPG)75 bilayers. Since the results obtained here agree very well with other methods, it is worthwhile to discuss in some detail the immersion depths of different parts of the lipid, which can be divided into headgroup and glycerol regions, as well as upper, central, and lower parts of the acyl chain.

DPPC77

a

For nomenclature, see Figure 1. bDistances from the bilayer center of acyl chain carbons are for the 16:0 (sn1) chain. cFor DMPC Cω3 = C12, Cω2 = C13, and CH3C14.

In all three bicelle types, the PC headgroup carbon atoms have approximately the same distance to the bilayer center which is in agreement with the observation that the P − N vector is almost perpendicular to the bilayer normal.71 The PC headgroups were found to be about 20 Å away from the bicelle center and from this value the bilayer thickness can be estimated to be 40 Å. Moreover, the thickness of the hydrocarbon region can roughly be estimated from the immersion depth of C2 to be 24 Å. Both the estimate of the thickness of the bilayer as well as the hydrocarbon region are in good agreement with X-ray and neutron diffraction studies that find DMPC bilayer thicknesses of 37−44 Å and a thickness of the hydrocarbon region of 26 Å.10−13 On average, acyl chains of E. coli lipids are 16 carbons long53 and, in this regard, are similar to 1,2-dipalmitoyl-sn-glycero-3phosphocholine (DPPC, 16:0−16:0 PC) or POPC (16:0− 18:1c9 PC) bilayers. These bilayers are about 2 Å thicker than DMPC bilayers. In our study, we find that the distance to the bilayer center of the PE and PG headgroups is on average 18 Å which is in good agreement with results obtained by SANS/ SAXS or in silico methods as can be seen in Table 5.75,76,78 However, because of the spatial resolution limits of our method (see below), the accuracy of the results is not sufficient to observe differences in membrane thickness between DMPC and E. coli lipid bilayers. SANS/SAXS studies of DMPG bilayers suggest that the distance to the bilayer center of the PG headgroup in anionic bicelles is underestimated in our study (see Table 5). Here we find that the DMPG headgroup is 14 Å away from the bilayer center whereas SANS/SAXS studies find 18 Å.75 On the other hand the estimate for the glycerol backbone (17 Å) agrees well with results from other methods (see Table 4),75 indicating that the PG headgroup in these bicelles is bent toward the surface of the bilayer. The immersion depths found for PG headgroups in E. coli bicelles is in agreement with MD simulations and SANS/ SAXS studies of POPG bilayers (see Table 5).75,79 Moreover, MD simulations find that the headgroup of POPG is almost parallel to the membrane surface with a downward tilt of PG-γ consistent with our observations.79 7665


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Table 5. Distances from the Bilayer Center of Headgroup Carbons in Anionic and E. coli Bicelles Compared to Results from Other Methodsa distance from the bilayer center/Å

a

anionic bicelles (this study)

E. coli bicelles (this study)

DMPG75

POPG75

POPG/POPE76

carbon atom

NMR

NMR

SANS/SAXS

SANS/SAXS

MD

PG-γ PG-β PG-α PE-β PE-α

14 ± 2 14 ± 2 15 ± 3 n.a. n.a.

15 ± 5 18 ± 4 21 ± 4 18 ± 4 19 ± 4

18 18 18 n.a. n.a.

21 21 21 n.a. n.a.

n.a. n.a. n.a. 18.2b 18.2b

For nomenclature, see Figure 1. bDistance of the phosphate group from the bilayer center.

Figure 4. Relative weights of A) 1 SBMF,2 and B) 1 SBMF,3 to the total value of 1 SBMF,1−3 upon variation of τloc and Sloc2. Colors represent 10%-intervals of the ratios. The other parameters are τbic = 200 ns, Slip2 = 0.48, τlip = 880 ps, and ωC = 2π × 150 MHz.

For the side chain modifications in E. coli (unsaturations and cyclopropanations) we observe distances from the bilayer center of 8 Å, i.e. at an intermediate distance between the bilayer center and the glycerol region of the lipid. In the E. coli AD93WT strain 16:1c and 18:1c acyl chains are approximately equally abundant (11−13%) and cyclopropanated chains (17:Δ) are somewhat more abundant (18%).49,53 For 16:1c and 17:Δ acyl chains, the modifications are located between C9 and C10; for 18:1c acyl chains, the unsaturation is located between C11 and C12.80 In MD simulations and SANS/SAXS studies, the unsaturation of POPC, i.e., 16:0−18:1c9 PC, is located at roughly 9 Å from the bilayer center, in good agreement with our results.11,68,74 Hyvönen et al. simulated bilayers of polyunsaturated and saturated lipids and analyzed the localization distribution of the side chain carbons.71 In their study, carbons C9−C12 of the diunsaturated sn-2 chain were found to be 8−11 Å away from the bilayer center. Moreover, carbon immersion depths in polyunsaturated lipids have a much broader distribution than in unsaturated lipids. This might be reflected by a large uncertainty in the carbon position of the E. coli lipid modifications observed here. Finally, the positions of the terminal carbons in our study seem to be somewhat too far away from the bicelle center when compared to bilayer simulations or SANS/SAXS studies. In particular, the methyl carbons are expected to be located closer to the bilayer center. As discussed below this discrepancy may be due to the simple structure of the spectral density function which does not include the rapid local dynamics of the terminal acyl chain carbons. Data Analysis and Lipid Dynamics. We employed two different forms of the spectral density function to describe the dynamics that lipids undergo in bicelles. In the simplest form the dynamics are described by a single correlation time, τr, which is usually identified with molecular, i.e. in this case bicelle,

tumbling. However, this assumes that no significant local motions exist.25 From the fitted constant C the correlation time τc can be calculated since all other constants in eq 2 are known. In the case of zwitterionic bicelles τc can thus be estimated from C = (0.043 ± 0.02) × 10−60 m6 s−1 as 12 ± 3 ps. Using Stokes’ law, the rotational correlation time of the bicelles can be obtained from their hydrodynamic radius81 which in turn can be estimated from measurements of translational diffusion coefficients.33 The hydrodynamic radius of small isotropic bicelles is about 6 nm33 corresponding to a rotational correlation time τbic of 200 ns which is many orders of magnitude larger than the τc estimate obtained from the data fit. This observation confirms the conclusion from previous studies that showed that the slow tumbling of bicelles has little influence on dipolar relaxation processes of lipid carbons.67 Therefore, 1 SBMF,1 in eq 5 is often neglected when dynamics of lipid carbons in bicelles are analyzed employing the extended model free approach. Importantly, it is not the bicelle tumbling that modulates the 13 C−e− vector reorientation but the additional degrees of motional freedom that exist in lipid bilayers. The spectral density function 1 SB (eq 3) constitutes a simplified description of the lipid dynamics in bicelles while 1 SBMF (eq 5) describes lipid motions in bicelles more comprehensively. In Figure 4, the contributions of the two dominating terms of 1 SBMF to its total value are displayed when τloc and Sloc2 are modified. For the specific case of zwitterionic bicelles analyzed here the weights of all three terms are given in Table S2 in the Supporting Information. The slow bicelle tumbling described by 1 SBMF,1contributes with less than 1% to the total value of 1 SBMF. In contrast, 1 SBMF,2, which describes the lipid dynamics, and 1 SBMF,3, which describes the 13C−1H bond dynamics, have significant contributions to 1 SBMF. For almost all lipid carbons, the largest contribution to 1 SBMF stems 7666


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The Journal of Physical Chemistry B from 1 SBMF,2, and therefore, the contributions of the dynamics of the entire lipid are most important. However, 1 SBMF,3 also contributes significantly to 1 SBMF even when local motions are restricted as in the case of the glycerol carbons. Yet, 1 SBMF,3 and thus local motions are more important when the local dynamics are unrestricted. Since lipids undergo a broad range of motions that may not be negligible in a PRE-analysis, we tested whether the use of 1 SBMF could improve the immersion depth estimates. This was done by employing order parameters and correlation times for DMPC lipids and 13C−1H bonds in the fitting procedure (see Table S1). Note that we thus did not explicitly consider the dynamics of the 13C−e− vector. As can be seen in Table 3, the major difference between using 1 SBMF and 1 SB in the analysis is in the immersion depth estimate for the glycerol carbons, which are found to be 26 Å instead of 15 Å away from the bilayer center. The immersion depth estimates for the glycerol carbons based on 1 SBMF is thus in clear contradiction to other studies (see Table 4). Moreover, there are also differences in the estimates for the methyl and the PC headgroup carbons. For the latter, the 1 SBMF based PRE-analysis resulted in a distance of 24 Å to the bilayer center; i.e., the immersion depth estimate of the PC headgroup deteriorated slightly (see Table 4). On the other hand, the immersion depths of the acyl chain carbons are somewhat closer to expected values. In particular, the extended analysis finds that the methyl carbons are located closest to the bilayer center, whereas Cω2 and Cω3 are further away. The improvements are, however, minor while the deterioration of the glycerol immersion depth estimate is large and significant. Yet, as has been pointed out in other studies, the estimates of Sloc2 and τloc are unreliable for the glycerol carbons since the fundamental assumption of time scale separation of the local and lipid motion is violated for slow and restricted motions.62,63 It has previously been concluded that Sloc2 is overestimated and τloc is underestimated for the glycerol carbons.49,63 Therefore, the underlying errors in the dynamic parameters of the glycerol carbons may yield an incorrect immersion depth estimate. On the other hand, the slight improvement in the estimate of the terminal acyl chain carbons may be due to the improved description of the lipid dynamics when using 1 SBMF in combination with reliable dynamic parameters of these carbons. In conclusion, a single correlation time spectral density function of the form of eq 3 is sufficient to yield good immersion depth estimates for all lipid carbons despite a wide range of local dynamics. Moreover, since the correlation time is fitted as a global variable, no assumptions concerning its magnitude need to be made. A more complicated spectral density function of the form of eq 5 may improve the estimate, however, this requires reliable information on the dynamic parameters of the lipid and the 13C−1H bonds. Standard deviations of the carbon positions were obtained from simulation of more than 200 bicelles and subsequent fitting of computed PREs to the experimental data by minimization of eq 6. Therefore, the distributions around the central carbon position do not reflect a measurement uncertainty but rather reflect a distribution of the carbon position inside the bicelle. The carbon distribution is thus a result of the fitting algorithm that randomly sets initial immersion depths of the spin-label and performs a least-squares fit based on these initial conditions. This approach is justified since carbon immersion depths are widely spread in bilayers, as evidenced by MD simulations (see Table 1) but also by the experimental data obtained here. For example, the PREs for 10-doxyl PC have very similar values for carbons g1 to C14 as can been seen in Figure 2C.

In addition to the structure of the spectral density function the theoretical description of the PRE has been simplified in other ways. By applying eq 2 we assume that the Solomon− Bloembergen equations (the inner-sphere relaxation model) are applicable in our case. In particular, this model requires that the paramagnetic species (the spin-labeled lipids) form a transient but sufficiently long-lived complex with the diamagnetic species (the lipids) during τM. If this assumption does not hold; i.e., if τM is too short, other more complex models that take the relative diffusion of the paramagnetic center with respect to the diamagnetic molecule into account (e.g., employing the outer-sphere relaxation model), need to be applied. In such models the distance dependence is not necessarily r−6.29 Although a more complicated distance law might apply here, the r−6 distance law has previously been applied successfully by others to similar systems.21,22 Moreover, the results obtained here are in good agreement with results from other methods justifying the approach a posteriori. Importantly, the immersion depth measurements rely on the accuracy of the underlying data. As can be seen in Figures 2, S3, and S5 the measurement uncertainties of the PREs are very high due to error propagation. We alleviate this problem by measurements of PREs in the presence of three or four different spin-labels. This leads to “blurry” but numerous constraints that need to be fulfilled simultaneously when minimizing eq 6 and thus counteract the effects of high measurement uncertainties of individual data points on the immersion depth estimate. Additionally, spectral overlap could lead to misinterpretation of the results. Carbon headgroup peaks of DMPC lipids overlap with headgroup peaks of DHPC-d22, which is located in the rim of the bicelle but neglected in our bicelle model. Similarly, all lipid and detergent glycerol carbon resonances (g1, g2, and g3) overlap. This may introduce a source of error in the estimate of immersions depths of PC headgroup and glycerol carbons. However, this error is most likely small or even negligible due to the r−6 distance dependence of the PRE. Because of the strong distance dependence only lipids in direct proximity to the spinlabel are significantly affected by its presence.21 Assuming that lipids and detergents partition into different regions of the bicelle, detergents are only affected by the spin-label if the spinlabeled lipid is located at the periphery of the bilayer. The contribution of DHPC to the measured PREs may thus be small. However, the fact that the PC headgroup carbons are found to be somewhat further away from the bilayer center than other headgroup carbon atoms might be a manifestation of peak overlap between detergent and lipid headgroup carbon atoms. Headgroup carbon resonances from E. coli phospholipids and from DMPG do not overlap with detergent resonances except for PG-β which overlaps slightly with g2. Since detergent acyl chains were deuterated, acyl chain carbon resonances from DHPC are broad multiplets and typically do not overlap with corresponding lipid resonances. Moreover, carbon atoms in unsaturations and cyclopronations show distinct resonances. Finally, the method relies on the accuracy of the a priori known immersion depths and immersion depths distributions of the spin-labels as given in Table 1. These immersion depths were derived from molecular dynamics simulations of the spin-labeled lipids in POPC bilayers.68 Since the acyl chains of the lipids of the E. coli strain used in this study are on average 16 carbon atoms long and about 30% of the lipids are unsaturated,53 it can be assumed that the membrane thickness of E. coli lipid bilayers is similar to that of POPC bilayers. Therefore, the spin-label immersion depths given in Table 1 are likely accurate in the case 7667


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The Journal of Physical Chemistry B of E. coli bicelles. In contrast, DMPC lipids are shorter than POPC lipids. In consequence, DMPC bilayers are about 2 Å thinner than POPC bilayers.11 This gives rise to an acyl chain mismatch between DMPC and the spin-labeled lipids. Therefore, the spin-label immersion depth estimates in Table 1 may not be fully transferable to zwitterionic bicelles. However, the deviations are expected to be small, compared to the accuracy of the method. This line of reasoning is reaffirmed by the fact that the carbon immersion depths obtained in this study agree well with results obtained by other methods. Deviations of up to 5 Å to other methods are observed for immersion depths measurements of DMPG headgroups in anionic bicelles (Table 5). A membrane thickness of 32.5 Å has been found for pure DMPG bilayers.75 They are thus substantially thinner than POPC bilayers (39.1 Å).11 The observed deviations between this study and other methods for headgroup immersion depth measurements of DMPG might thus be due to inaccurate assumptions on the immersion depths of the spin-labels in bilayers that contain a significant amount of DMPG. The comparison of our data with experimental and simulated data suggests that despite the simplifications of the underlying theory and possible inaccuracies in the presupposed spin-label positions, we obtained results that are in very good qualitative agreement with other experimental methods and MD simulations. Deviations between the immersion depths observed here and published results do not exceed 3−5 Å, which may then be interpreted as the resolution limit of the method presented here. With such a resolution, it is possible to distinguish the immersion depth of different regions of the lipids inside the bilayer, i.e. the headgroup and the glycerol regions as well as the upper, central, and terminal regions of the acyl chain.

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AUTHOR INFORMATION

Corresponding Author

*(J.L.) E-mail: [email protected]. ORCID

Jobst Liebau: 0000-0003-4057-6699 Lena Mäler: 0000-0002-9464-4311 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Philipp Zuber for his help in conducting the PRE experiments. We thank Weihua Ye for providing us with R1 and NOE data of DMPC in galactolipid bicelles. This work was supported by the Swedish Research Council (Contract No. 6212014-3706).

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ABBREVIATIONS 5/10/16-doxyl PC, 1-palmitoyl-2-stearoyl-(5/10/16-doxyl)-snglycero-3-phosphocholine; DHPC-d22, tail-deuterated 1,2-dihexanoyl-sn-glycero-3-phosphocholine; DMPC, 1,2-dimyristoyl-snglycero-3-phosphocholine; DMPG, 1,2-dimyristoyl-sn-glycero3-phosphoglycerol; DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; MD, molecular dynamics; PC, phosphatidylcholine; PG, phosphatidylglycerol; PE, phosphatidylethanolamine; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; POPE, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; POPG, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol; PRE, paramagnetic relaxation enhancement; SANS, small-angle neutron scattering; SAXS, small-angle X-ray scattering; TEMPO-PC, 1,2-dipalmitoyl-sn-glycero-3-phospho(tempo)choline

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CONCLUSIONS We measured the immersion depths of lipid carbons in three different bicelle systems, including bicelles made from natural E. coli lipid extract, by measuring PREs of the longitudinal relaxation rate R1 in the presence of three or four different doxylated lipid spin-labels. To do so, PREs were computed theoretically in a simple bicelle model employing a r−6 distance dependence of the PRE. The distance between the computed and experimental PREs were then minimized by varying the carbon immersion depths in the simulated bicelles. We find that the localizations of lipid carbons inside the membrane observed here are in good agreement with studies that employed other methods and sufficiently accurate to distinguish the membrane immersion depths of different regions of the lipid despite rather crude theoretical assumptions. These involve among others a simple form of the spectral density function, which describes the lipid dynamics by a single correlation time. The method presented here allows to quantify immersion depths of individual carbon atoms in lipid bilayers by solution state NMR. It may be extended to investigations of membrane protein localization within biological membranes potentially employing realistic membrane mimetic systems made from, e.g., E. coli phospholipid extract.

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atoms in anionic and E. coli bicelles, Q-factors of the immersion depth fits for anionic and E. coli bicelles, dynamic parameters for DMPC in bicelles enriched with galactolipids, weights of the terms of the extended spectral density function for DMPC carbon atoms, and PREs (Γ1) and Q-factors for zwitterionic bicelles calculated using the extended spectral density function (PDF)

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.7b05822. 13 C and 31P spectra of zwitterionic, anionic and E. coli bicelles, measured and calculated PREs (Γ1) of carbon 7668


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