Solubilization of lipids and lipid phases by the styrene–maleic acid copolymer
Solubilization of lipids and lipid phases by the styrene-maleic acid copolymer
Juan J. Dominguez Pardo 0 1 2
Jonas M. Dörr 0 1 2
Aditya Iyer 0 1 2
Ruud C. Cox 0 1 2
Stefan Scheidelaar 0 1 2
Martijn C. Koorengevel 0 1 2
Vinod Subramaniam 0 1 2
J. Antoinette Killian 0 1 2
0 Present Address: Vrije Universiteit Amsterdam , De Boelelaan 1105, 1081 Amsterdam , The Netherlands
1 Nanoscale Biophysics Group, FOM Institute AMOLF , Science Park 104, 1098 Amsterdam , The Netherlands
2 Department of Chemistry, Faculty of Science , Membrane Biochemistry and Biophysics , Bijvoet Center for Biomolecular Research , Padualaan 8, 3584 Utrecht , The Netherlands
A promising tool in membrane research is the use of the styrene-maleic acid (SMA) copolymer to solubilize membranes in the form of nanodiscs. Since membranes are heterogeneous in composition, it is important to know whether SMA thereby has a preference for solubilization of either specific types of lipids or specific bilayer phases. Here, we investigated this by performing partial solubilization of model membranes and analyzing the lipid composition of the solubilized fraction. We found that SMA displays no significant lipid preference in homogeneous binary lipid mixtures in the fluid phase, even when using lipids that by themselves show very different solubilization kinetics. By contrast, in heterogeneous phase-separated bilayers, SMA was found to have a strong preference for solubilization of lipids in the fluid phase as compared to those in either a gel phase or a liquid-ordered phase. Together the results suggest that (1) SMA is a reliable tool to characterize native interactions between membrane constituents, (2) any solubilization preference of SMA is not due to properties of individual lipids but rather due to properties of the membrane or membrane domains in which these lipids reside and (3) exploiting SMA resistance rather than detergent resistance may be an attractive approach for the isolation of ordered domains from biological membranes.
Lipid-protein interactions; Nanodiscs; Styrene-maleic acid; SMALP; SMA-resistant membrane (SRM); Lipid rafts
-
In recent years, the styrene–maleic acid (SMA) copolymer
has evolved as an important tool for isolation and
characterization of membrane proteins [for review, see (Dörr et al.
2016)]. SMA has been shown to solubilize biological
membranes in the form of nanodiscs allowing the isolation of
membrane proteins directly from their native environment
without the need for detergent (Long et al. 2013; Gulati
et al. 2014; Jamshad et al. 2015; Prabudiansyah et al. 2015;
Swainsbury et al. 2014; Dörr et al. 2014). The small size of
these so-called “native nanodiscs” enables their
characterization by a variety of biophysical approaches (Swainsbury
et al. 2014; Dörr et al. 2014, 2016; Orwick et al. 2012;
Jamshad et al. 2014; Orwick-Rydmark et al. 2012; Vargas et al.
2015) Furthermore, the presumed preservation of the
annular lipid environment helps to maintain the stability of the
embedded proteins and thereby allows the use of SMA as a
convenient tool to study preferential lipid–protein
interactions, simply by analyzing the lipid composition of purified
protein-containing nanodiscs and comparing it with that of
the native membrane (Swainsbury et al. 2014; Dörr et al.
2014; Prabudiansyah et al. 2015).
For unambiguous analysis of preferential lipid–protein
interactions using SMA, it is however of crucial
importance to know whether or not SMA by itself exhibits any
lipid preference during solubilization. This can be
conveniently investigated by employing synthetic model membrane
systems, which allow highly systematic variation of lipid
composition. Solubilization of model membranes by SMA
results in the formation of styrene–maleic acid lipid
particles (SMALPs), which have similar sizes and properties as
membrane protein-containing (native) nanodiscs (Dörr et al.
2016). Using such model systems, it has been shown that
the interaction of SMA with membranes strongly depends
on lipid composition, with the kinetics of solubilization
being modulated by, e.g., surface charge, lipid packing and
lipid chain length (Scheidelaar et al. 2015). This would
suggest that SMA might exhibit a lipid preference toward
solubilization. However, experiments in which model
membranes of an Escherichia coli total lipid extract were
partially solubilized showed that the SMA-solubilized fraction
exhibits no significant enrichment in specific lipid species
(Scheidelaar et al. 2015). Together these results suggest that
SMA is promiscuous and that solubilization is determined
by overall properties of the membrane rather than by
properties of individual lipids. This was supported by a recent
study using 31P NMR (Cuevas Arenas et al. 2016).
So far, the lipid mixtures that have been used to study
preferential solubilization by SMA represent only a few
selected homogeneous lipid mixtures in the fluid phase,
and no systematic studies have been reported yet on a
possible lipid preference of SMA. Also, whether SMA exhibits
any preference in heterogeneous membranes that exhibit
domain formation and that arguably are biologically more
relevant than homogeneous fluid bilayers has not been
investigated.
To obtain insight into these matters, we here set out to
investigate to what extent preferential solubilization of
lipids by SMA occurs in simple binary lipid systems
forming a single homogeneously mixed fluid phase and in
heterogeneous phase-separated membranes exhibiting
coexistence of a fluid phase with either gel or liquid-ordered
phase. In order to maximize our “window” for monitoring
any potential preferences of the polymer, the following
strategy was employed. First, combinations of lipids were
selected that on their own would have very different SMA
solubilization kinetics. Second, to achieve partial
solubilization short incubation times of 1 h were used thereby
avoiding full equilibration of the system. This required
adjustment of the concentration of SMA for each system
individually in order to obtain sufficient material for
reliable analysis. Third, multilamellar vesicles (MLVs) were
chosen as the lipid system, which has the following
advantages: (1) MLVs provide a large accessible surface area,
which diminishes the chance of “all or nothing” effects
that may occur for small vesicles, i.e., full solubilization
of some membranes and no solubilization of others, (2) by
employing these larger structures any potential curvature
effects are avoided, and (3) the use of these larger
structures facilitates the separation of solubilized and
non-solubilized material by centrifugation.
The results show that SMA is indeed highly
promiscuous with respect to solubilization of lipid species in
homogeneous fluid bilayers, but that there is a clear preference
for solubilization of the fluid phase in phase-separated
bilayers with either a gel phase or a fluid phase. We will
discuss the implications of these findings regarding the
general applicability of SMA as a tool to determine
preferential lipid–protein interactions. We will also discuss the
use of SMA for the isolation of SMA-resistant membranes
(SRMs) as an alternative to conventionally studied
detergent-resistant membranes (DRMs).
Materials and methods
All lipids were purchased from Avanti Polar Lipids
(Alabaster, AL). The used lipids were
1,2-dioleoyl-snglycero-3-phosphocholine (di-18:1 PC);
1,2-dioleoylsn-glycero-3-phospho(1′-rac-glycerol) (di-18:1 PG);
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (di-18:1 PE);
1,2distearoyl-sn-glycero-3-phosphocholine (di-18:0 PC);
1,2dimyristoleoyl-sn-glycero-3-phosphocholine (di-14:1 PC);
1,2-di-(9Z-hexadecenoyl)-sn-glycero-3-phosphocholine
(di-16:1 PC);
1,2-di-(11Z-eicosenoyl)-sn-glycero-3-phosphocholine (di-20:1 PC);
1,2-dierucoyl-sn-glycero-3-phosphocholine (di-22:1 PC);
1-palmitoyl-2-oleoyl-sn-glycero3-phosphocholine (16:0/18:1 PC); brain sphingomyelin
(bSM); cholesterol; 23-(dipyrrometheneboron
difluoride)24-norcholesterol (Top-Fluor-cholesterol);
1,2-dioleoyl-snglycero-3-phosphoethanolamine-N-(lissamine rhodamine B
sulfonyl) (rhodamine-PE).
Xiran 25010, a styrene–maleic anhydride copolymer
with a molar ratio of styrene-to-maleic anhydride of 3:1
and an average molecular weight of 10 kDa, was a kind gift
from Polyscope (Geleen, The Netherlands). Xiran 25010
(anhydride copolymer) was converted to the acid form by
hydrolysis under base-catalytic conditions as detailed
elsewhere (Scheidelaar et al. 2015). All other chemicals used
were from Sigma Aldrich (St. Louis, MO).
Preparation of multilamellar vesicles (MLVs)
Phospholipid stock solutions in chloroform/methanol (9:1
v/v) were mixed in predetermined ratios, and the solvent
was removed under a stream of N2. The resulting lipid film
was dried in a desiccator under vacuum for at least 1 h.
MLVs were obtained by hydrating the lipid films with
solubilization buffer (50 mM Tris–HCl, 150 mM NaCl, pH 8.0).
The samples were then subjected to ten freeze–thaw cycles,
each consisting of 3 min of freezing in liquid N2 (−196 °C)
and 3 min of thawing in a water bath at 50 °C, well above
the gel-to-fluid phase transition temperature (Tm) of the
lipids. For vesicles containing di-18:0 PC [Tm = 56 °C
(Marsh 2013; Lewis et al. 1987)], the water bath was kept
at 60 °C to ensure membrane fluidity.
Turbidimetry experiments
The solubilization of MLVs was monitored by
turbidimetry, using a Lambda 18 spectrophotometer (PerkinElmer,
Waltham, MA) as described previously (Scheidelaar et al.
2015). Briefly, 700-µl aliquots of 0.5-mM dispersions of
MLVs in solubilization buffer were transferred to a quartz
cuvette and equilibrated at the desired temperature for
10 min. Next, different amounts of SMA were added as
detailed in the legends of the corresponding figures, and
the solubilization kinetics were followed at a fixed
wavelength of 350 nm by monitoring the decrease of the
apparent absorbance. Absorbance values were recorded every
0.4 s.
Analysis and quantification of the lipid composition
of solubilized fractions
Partial solubilization of vesicles
To achieve partial solubilization while still obtaining
enough lipid material for further analysis, the SMA
concentration was tuned for each lipid mixture individually.
Accordingly, 700-μl aliquots of 0.5-mM dispersions of
MLVs in solubilization buffer were supplemented with an
amount of SMA that led to a decrease in apparent
absorbance to approximately 50–60% after 1 h of incubation.
Vesicles containing liquid-ordered domains required higher
amounts of SMA for sufficient solubilization because of
their higher solubilization resistance. Detailed incubation
conditions for each sample are specified in the legends of
the corresponding figures.
After 1 h of incubation with SMA, the samples were cooled
down on ice and then transferred to a pre-chilled
ultracentrifuge. The non-solubilized material was removed
by centrifugation at 115,000×g for 1 h at 4 °C, and the
supernatant, containing the solubilized lipid material, was
collected. The lipids from the supernatant and from an
aliquot of non-treated MLVs were extracted according to the
method of Bligh and Dyer (Bligh and Dyer 1959) (see
Supporting Information) prior to analysis.
Lipid analysis and quantification
The procedure for lipid analysis and quantification was
selected depending on the lipid composition of the
membrane. Lipids with different headgroups were separated
by thin layer chromatography (TLC). Quantification was
then achieved by densitometric analysis after copper
staining (Dörr et al. 2014; Swainsbury et al. 2014).
Phospholipids with the same headgroup but with unsaturated acyl
chains of different length were separated by reverse-phase
TLC. After iodine staining, each spot was scraped off, and
the amount of phosphate was determined by the method
of Rouser (Rouser et al. 1970). For phospholipids with the
same headgroup but with acyl chains differing in degree of
unsaturation, reverse-phase TLC did not provide sufficient
separation. These samples were therefore quantified by gas
chromatography after esterification of the fatty acids (de
Smet et al. 2012; Dörr et al. 2014). For detailed
experimental descriptions, see Supporting Information.
Transmission electron microscopy
Size characterization of the SMALPs present in the
supernatant fractions resulting from turbidimetry experiments
was performed by transmission electron microscopy. To
this end, copper grids were prepared following the
carbon flotation technique. Briefly, samples were diluted with
solubilization buffer to a lipid concentration of 0.5–1 mM,
and small aliquots were adsorbed on carbon-coated mica.
The mica was then transferred to a staining solution
containing 2% (w/v) sodium silico tungstate, causing
detachment of the carbon film. Subsequently, a copper grid was
placed on top of the detached carbon that was recovered
and dried under air flow. Images were taken under low dose
conditions at a nominal magnification of 49,000 with a T12
electron microscope (FEI, Hillsboro, OR) at an operating
voltage of 120 kV using an ORIUS SC1000 camera (Gatan,
Inc., Pleasanton, CA). The average size of the SMALPs
was estimated manually from 16 well-defined individual
particles randomly located through the image based on
their maximum diameter using Adobe Illustrator software
(San Jose, CA). This procedure was used to avoid potential
artifacts such as stain-induced particle aggregation or
inhomogeneous particle staining (Zhang et al. 2011; Wan et al.
2011; Scheidelaar et al. 2015).
Fluorescence microscopy imaging was performed at
room temperature using a Nikon A1 confocal microscope
(Tokyo, Japan) equipped with a Perfect Focus system.
Supported lipid bilayers (SLBs) were prepared in a
custombuilt chamber following the vesicle fusion procedure (see
Supporting Information). Solubilization of SLBs by SMA
was assessed under a continuous flow of solubilizing agent
solution. Images were taken before addition of SMA and
after 5 min of incubation using a 100× oil immersion
1.49NA objective (Nikon) under identical conditions of laser
power and gain for all samples. Top-Fluor cholesterol and
rhodamine-PE were imaged sequentially using a 488- and
561-nm laser, respectively, to avoid spectral cross-talk. The
images were acquired with a resolution of 512 × 512 pixels
(pixel size 0.41 × 0.41 μm).
Fluorescence intensities were quantified from intensity
histograms using NIS Elements software (Nikon). Intensity
values are expressed as an average of the intensities
calculated from five different snapshots randomly picked from
the planar bilayer. A representative video of the
solubilization process can be found in the Supporting Information
(Video S1).
It was previously demonstrated in model membrane
systems that the lipid headgroup and acyl chain
composition are important determinants for the kinetics of SMA
solubilization (Scheidelaar et al. 2015). Here, we
investigated whether two lipids that exhibit very different
solubilization kinetics will be selectively solubilized by SMA
when homogeneously mixed in a lipid bilayer. For this we
selected mixtures of di-18:1 PC as “host” lipid with an
equimolar amount of different “guest” lipids. The choice
of these guest lipids was motivated by our previous
observations (Scheidelaar et al. 2015) that (1) lipids with short
chains are solubilized faster than lipids with longer acyl
chains, most likely as a consequence of the lower number
of van der Waals interactions between neighboring chains;
(2) bilayers containing negatively charged lipids exhibit
much slower solubilization kinetics than bilayers of
zwitterionic lipids, presumably due to electrostatic repulsion by
the negative charge of the polymer; (3) bilayers
containing lipids in the gel phase, cone-shaped lipids or
unsaturated lipids are solubilized more slowly than bilayers
containing cylindrical lipids or saturated lipids in the fluid
phase. These latter effects were ascribed to differences in
the packing density of the acyl chains, with tighter
packing hindering the insertion of the polymer and subsequent
solubilization.
To obtain insights into a possible selectivity of SMA for
certain lipids, an approach of partial solubilization of MLVs
was used, as illustrated in Fig. 1a for a mixture of di-18:1
PC with the anionic lipid di-18:1 PG, which is known to
form homogeneously mixed bilayers (Marsh 2013; Nibu
et al. 1995). The soluble fraction, after incubation with SMA
for 1 h, was subjected to electron microscopy (EM) imaging
(Fig. 1b) and lipid composition analysis by TLC (Fig. 1c).
The EM data (Fig. 1b) show a homogeneous distribution of
particles of 6–8 nm size (Table 1), which is at the lower end
of the range of commonly reported dimensions of around
10 nm [see, e.g., (Scheidelaar et al. 2015; Jamshad et al.
2014; Orwick et al. 2012)]. Lipid composition analysis of
the solubilized fraction after SMA incubation revealed that
PC and PG are present in a similar molar ratio as in the
initial vesicles (Fig. 2a), indicating non-selective solubilization
of both lipids. Considering the electrostatically unfavorable
interaction of SMA with negatively charged lipids
(Scheidelaar et al. 2015), this result is rather surprising and suggests
that SMA does not perturb the bilayer homogeneity.
Similar experiments were performed with other
homogeneous lipid mixtures in the fluid phase. The results are
summarized in Fig. 2a and Table 1, while original
solubilization traces and representative EM micrographs can
be found in Figure S1. When di-18:1 PC was mixed with
the cone-shaped lipid di-18:1 PE, again it was found that
SMA does not show a lipid preference (Fig. 2a), despite
the slower kinetics of solubilization of PC/PE as
compared to pure PC bilayers (Scheidelaar et al. 2015). This
non-selective solubilization is in line with results from a
recent study of a very similar lipid system (Cuevas Arenas
et al. 2016). A different result was obtained for mixtures of
lipids with varying acyl chain length, where a small
preference was observed for solubilization of di-14:1 PC over
di-18:1 PC (Fig. 2a). To elucidate whether this preference
might be related to hydrophobic mismatch effects, we also
tested mixtures in which di-14:1 PC was kept as the shorter
lipid, while the length difference between the lipid
components was either increased or decreased (Figure S2A). In
all cases, the results showed a similar small preference for
di-14:1 PC, suggesting that this is an artifact related to a
particular feature of di-14:1 PC, perhaps being more easily
extracted from the membrane because of its short
unsaturated acyl chains. This hypothesis is supported by the
results obtained from partial solubilization of di-18:1 PC/
di-22:1 PC membranes (Figure S2B), where the solubilized
fraction has a similar lipid composition as the initial
vesicles. Finally, a mixture of di-18:1 PC with di-18:0 PC was
tested under conditions where both lipids were in the fluid
phase. This was achieved by raising the incubation
temperature to 60 °C, above the gel-to-liquid crystalline phase
transition temperature of di-18:0 PC. Here again no
preference for either lipid species was observed (Fig. 2a).
Fig. 1 Partial solubilization of
vesicles by SMA. a Kinetics of
SMA solubilization of MLVs
composed of an equimolar
mixture of di-18:1 PC and
di-18:1 PG (0.5 mM lipid,
SMA-to-lipid mass ratio of
0.64) at 25 °C. Data are shown
as normalized optical density at
350 nm. b Visualization of the
SMALPs from the supernatant
by negative-stain
transmission electron microscopy. c
Thin layer chromatography
analysis of the lipid
composition of lipids extracted from
non-treated MLVs as well as
the soluble fraction after partial
solubilization by SMA
Table 1 Nanodisc size characterization based on analysis of EM data
Lipid mixture (1:1, M)
Incubation temperature (°C)
di-18:1 PC/di-18:1 PG
di-18:1 PC/di-18:1 PE
di-18:1 PC/di-14:1 PC
di-18:1 PC/di-18:0 PC
di-18:1 PC/di-18:0 PC
For all the solubilized fractions corresponding to Fig. 2,
the formed SMALPs appeared to have a rather similar size in
the range of 6–10 nm as visualized by EM imaging (Fig. 1b,
Figure S1) and as quantified in Table 1. Previously it was
reported that the use of relatively low SMA concentrations
might result in the formation of larger particles (Vargas et al.
2015; Zhang et al. 2015). However, in our case the different
populations of SMALPs were found to be fairly small with a
relatively uniform size distribution, despite conditions of
relatively low SMA concentrations (SMA-to-lipid mass ratio of
0.3–1.3). Importantly, similar particle sizes were found under
conditions of using a higher SMA-to-lipid ratio, longer
incubation times and higher lipid concentrations, which allowed
characterization of the particles by both EM and dynamic
light scattering (Figure S2, Table S1). Together these data
support the validity of our partial solubilization approach.
Overall, the data show that SMA is highly
promiscuous with respect to solubilization of lipid species when
these are present as homogeneously mixed bilayers in the
fluid phase. Whether preferences of SMA solubilization do
occur in bilayers with a heterogeneous lipid distribution
was investigated next.
A heterogeneous lipid bilayer can easily be obtained in
mixtures of lipids with unsaturated (low Tm) and
saturated (high Tm) acyl chains by lowering the temperature
well below Tm of the saturated lipid. For instance, in the
above-described equimolar mixture of di-18:1 PC and
di-18:0, lowering the temperature to 25 °C promotes a
situation where gel and fluid (liquid–crystalline) phases
coexist (Marsh 2013). Under these conditions, SMA shows
a strong preference toward solubilizing the fluid phase,
which is mainly constituted by di-18:1 PC (Fig. 2b). This
result is in accordance with the much faster solubilization
kinetics of lipids in the fluid phase as compared to lipids in
the gel phase (Scheidelaar et al. 2015; Cuevas Arenas et al.
2016). For bilayers exhibiting phase coexistence, the lipid
preferences of SMA under conditions of partial
solubilization thus do appear to reflect the differences in
solubilization kinetics between the lipids in their respective phases.
SMA preferentially solubilizes the fluid
liquid‑disordered matrix upon incubation
with membranes containing liquid‑ordered domains
The resistance of gel-phase lipids against
solubilization by SMA raises the question whether this is a general
phenomenon for phases in which the lipids exhibit a high
degree of order. This was first tested by adding SMA to
a binary mixture of brain sphingomyelin (bSM) and
cholesterol that forms a liquid-ordered (Lo) phase (Sankaram
Fig. 2 Solubilization preference of SMA in binary lipid systems with
different properties assessed by lipid composition analysis after
partial solubilization. a Equimolar mixtures of the zwitterionic
unsaturated di-18:1 PC (“host”, orange) with different guest lipids (green)
under conditions of phase homogeneity. From left to right: anionic
di-18:1 PG, cone-shaped di-18:1 PE, short chain di-14:1 PC and
saturated di-18:0 PC. Respective SMA-to-lipid mass ratios at 0.5 mM
lipid were 0.64, 1.31, 0.27 and 0.13. Phase homogeneity for di-18:1
and Thompson 1990; de Almeida et al. 2003). Addition of
an amount of SMA that is generally sufficient to rapidly
solubilizes homogeneous bilayers in the fluid phase
(SMAto-lipid mass ratio of 3.5) did not lead to any decrease in
apparent absorbance after 1 h for this system (Figure S4),
and neither did increasing the SMA concentration or
prolonging incubation times (data not shown), indicating a
very poor solubilization efficiency of SMA for lipids in
the Lo phase. These results resemble those reported for the
non-ionic detergent Triton X-100 (TX-100), for which the
Lo phase shows a well-described detergent resistance [see,
e.g., (El Kirat and Morandat 2007; Veiga et al. 2001; Rinia
et al. 2001; Patra et al. 1999; Sot et al. 2002; London and
Brown 2000)].
The solubilization potential of SMA was further
investigated in an equimolar ternary lipid mixture of di-18:1
PC, bSM and cholesterol. Over a wide temperature range,
bilayers of such composition exhibit phase separation
containing Lo domains enriched in sphingomyelin and
cholesterol that coexist with a fluid liquid-disordered (Ld)
matrix enriched in di-18:1 PC (Veatch and Keller 2003;
Marsh 2013; de Almeida et al. 2003). As shown from the
TLC results in Fig. 3a and as quantified in Fig. 3b, the lipid
material solubilized from these membranes after incubation
with SMA at 25 °C is clearly enriched in di-18:1 PC, while
it is depleted in bSM and cholesterol in approximately
equimolar amounts. At 4 °C, the SMA-solubilized fraction
PC/di-18:0 PC was achieved by elevating the temperature to 60 °C,
above Tm of di-18:0 PC (Tm = 56 °C) (Marsh 2013; Lewis et al.
1987). b Equimolar mixture of di-18:1 PC and di-18:0 PC under
conditions of phase separation at 25 °C (SMA-to-lipid mass ratio 1.27).
Cartoons show the schematic bilayer organization before addition of
SMA. Error bars represent the standard deviation of three
independent experiments
has a rather similar lipid composition as at 25 °C, while at
37 °C the solubilized fraction resembles the non-treated
case more closely (Fig. 3b). These results are consistent
with a preferential solubilization of the Ld phase over the
Lo phase by SMA at lower temperatures, which may be
ascribed to tight packing and preferential SM–cholesterol
interactions that cause co-segregation from the fluid phase
(Veiga et al. 2001; Sankaram and Thompson 1990; Patra
et al. 1999; Veatch and Keller 2003). Indeed, in the absence
of cholesterol, an equimolar mixture of bSM and di-18:1
PC was found to be solubilized in equimolar amounts of
both lipids (Figure S5), demonstrating the large effect of
cholesterol on lipid organization. At 37 °C, both cholesterol
and bSM are more readily solubilized, likely to be related
to the beginning of a gradual liquid ordered-to-fluid phase
transition (Lichtenberg et al. 2005; McMullen et al. 2004).
Based on our findings of non-preferential SMA
solubilization in homogeneous bilayers (Fig. 2a), it is likely that also
this phase-separating ternary lipid mixture will be
solubilized without a (strong) preference in case it exists in a
homogeneous fluid phase. Similar results showing a high
predisposition of the SMA copolymer to solubilize the Ld
phase over the Lo phase were obtained for a
phase-separating ternary lipid mixture of 16:0/18:1 PC, bSM and
cholesterol (Figure S6).
To gain more insight into the mode of action of SMA
in phase-separated bilayers, we performed additional
Fig. 3 Lipid composition analysis after partial solubilization of
MLVs composed of an equimolar ternary lipid mixture of di-18:1
PC, bSM and cholesterol by SMA. The inset shows a simplified
schematic cartoon representation. a TLC plate with lipids extracted
from non-treated vesicles and from the soluble fraction after
incubation with SMA at 25 °C. b Quantification of lipid composition shown
experiments where the solubilization of di-18:1 PC/bSM/
cholesterol membranes was monitored using
fluorescence microscopy. For these experiments we used
supported lipid bilayers (SLBs) that were supplemented with
a small amount of lipid-derived fluorescent dyes that
partition selectively into the Lo or Ld phase (Klymchenko and
Kreder 2014). At room temperature, the SLBs showed a
clear phase separation, with Lo domains varying in size
from 0.1–2 μm (Fig. 4a). After 5 min of incubation with
0.1% (w/v) of SMA, the fluorescence intensity of the
Ld probe dropped by more than 50%, while the
fluorescence emitted by the Lo probe decreased only marginally
(Fig. 4b). When the SMA concentration was increased to
0.5% (w/v) only background levels of Ld fluorescence
could be detected, while the Lo fluorescence was still at
approximately 40% of the initial intensity. Importantly,
no further decrease in Lo fluorescence was observed when
the SMA concentration was further increased to 1% (w/v)
(Fig. 4b, see also Video S1) or when the sample was
allowed to further incubate with the SMA solution for
several hours (data not shown). Thus, the Ld phase enriched
in di-18:1 PC is efficiently solubilized by SMA, while the
Lo domains show a high resistance against solubilization,
which is in agreement with the experiments performed with
vesicles at the same temperature.
The experiments described in this study reveal new insights
into the process of membrane solubilization by SMA and
how it depends on physicochemical properties of individual
as mol% lipid (color coding consistent with cartoon) for non-treated
vesicles as well as the solubilized fractions after the incubation with
SMA (0.5 mM lipid, SMA-to-lipid mass ratio of 3.1) at different
temperatures. Error bars represent the standard deviation of three
independent experiments
lipids and those of the membrane or membrane domains
they reside in. Here, we will discuss our findings and the
implications for the use of SMA as a tool to (1) study lipid–
protein interactions and (2) isolate ordered domains from
biological or model membranes.
SMA as a tool to study lipid–protein interactions
To obtain insight into whether SMA by itself has any
preference for solubilization of specific lipids, we performed
partial solubilization experiments on binary lipid mixtures.
The results strongly suggest that under conditions of phase
homogeneity there is no significant preference of SMA to
solubilize any glycerophospholipid species. This is
irrespective of differences in solubilization kinetics of the
individual lipids upon changing properties such as headgroup
charge, lipid shape, acyl chain saturation or acyl chain
length. A potential exception is di-14:1 PC, which was
found to be incorporated into SMALPs with a slight
preference, probably due to its short and unsaturated acyl chains.
Importantly, the observed full promiscuity of SMA in
solubilizing homogeneous fluid bilayers suggests that SMA
by itself does not perturb the bilayer homogeneity. Thus,
our findings support the validity of the use of SMA to
study preferential lipid–protein interactions of membrane
proteins that are extracted from biological membranes
(Swainsbury et al. 2014; Dörr et al. 2014; Prabudiansyah
et al. 2015). Here, a snapshot view of the interplay of lipids
and proteins in biological membranes can be obtained,
provided that the proteins reside in a fluid lipid environment.
However, biological membranes in general are
heterogeneous and may contain domains that are more
Fig. 4 Preferential solubilization in supported lipid bilayers
composed of an equimolar ternary lipid mixture of di-18:1 PC, bSM and
cholesterol. a Fluorescence microscopy images are shown as merged
(left column) and single channels (green top-fluor-cholesterol, middle
column; red: rhodamine-PE, right column) for non-treated samples
and after incubation times of 5 min with different amounts of SMA
in solubilization buffer. The scale bars correspond to 10 µm. b
Quantification of the fluorescence intensity in the images from (a). Dashed
lines are depicted to guide the eye. All experiments were performed
at room temperature. Error bars represent the standard deviation of
the fluorescence intensity of five snapshots randomly picked from the
planar bilayer
ordered (Simons and Ikonen 1997; Brown and Rose 1992;
Schroeder et al. 1994). Our experiments with
phase-separating lipid bilayers show that distinct solubilization
preferences of SMA do arise under conditions of phase
coexistence of a fluid phase with either a gel phase or a Lo phase.
In both cases, the soluble fractions consisted almost
exclusively of lipids that were in the fluid phase. For gel phases
it was previously postulated that it is the tight packing of
the chains that is responsible for the poor solubilization
yield, because it increases the energetic barrier for SMA
molecules to penetrate into the bilayer core (Scheidelaar
et al. 2015; Cuevas Arenas et al. 2016). The same
explanation would hold for the Lo phase, because it displays a
similarly high degree of order of the acyl chains (Ipsen
et al. 1987). Together these results demonstrate that lipid
packing plays a major role in the resistance against
solubilization by SMA. What are the implications of these results
for the use of SMA for the investigation of preferential
lipid–interactions for proteins that reside in either gel phase
domains or liquid-ordered domains? Obviously, such
proteins will not easily be solubilized into SMALPs. However,
they may be isolated as insoluble domains instead, as will
be further discussed below. Although analysis of the lipid
environment in such a case will not provide a snapshot of
the immediate lipid environment, it may nevertheless
provide relevant information on the lipid composition of the
domains in which the protein resides.
SMA as a tool to isolate ordered domains
The clear preference of SMA to solubilize the fluid phase
under conditions of phase coexistence holds promise for
applications for the isolation of SMA-resistant membrane
(SRM) domains. On the one hand, these could be applied
to domains with a very high protein density, as was recently
demonstrated by experiments in which SMA was used to
prepare thylakoid membrane fractions that are enriched
in specific photosystem complexes (Bell et al. 2015). On
the other hand, they could involve approaches similar to
those exploiting detergent resistance of certain membrane
domains.
Resistance against detergent solubilization is a
wellknown phenomenon in membrane research, which has been
used extensively to prepare DRMs from biological
samples (Cerneus et al. 1993; Hanado et al. 1995; Brown and
Rose 1992; Schroeder et al. 1994). In particular, TX-100
resistance at low temperatures has been exploited for the
isolation of DRM fractions from mammalian plasma
membranes. These DRM fractions have been associated to
socalled “lipid rafts,” which are postulated to be specific
membrane domains that are enriched in
(glyco)sphingolipids, cholesterol and specific proteins and that have
important roles in membrane function (Schroeder et al. 1994).
The basis of their detergent resistance is ascribed to the
ordered nature of the lipid chains in these domains (Simons
and Ikonen 1997; Schroeder et al. 1994). Our results with
model membranes suggest that SMA may be used in a
similar way as conventional detergent to isolate highly ordered
membrane domains in the form of SRMs.
This raises the question of how the two approaches to
isolate ordered domains from biological membranes would
compare. DRMs are unlikely to have the same composition
as postulated natively occurring lipid rafts in the plasma
membrane at physiological temperature (Heerklotz 2002;
Lichtenberg et al. 2005). One reason for this is that
conditions for DRM isolation usually include low temperatures,
which will promote phase separation and thereby may
cause further deviation from the composition of lipid rafts
as they may occur at physiological temperature.
Furthermore, by partitioning into the membrane, TX-100 shifts the
thermodynamic equilibrium of phase separation (Heerklotz
2002) and thus likely affects the composition of lipid rafts
that are isolated in DRMs. Alternatively, TX-100 has been
postulated to increase the sizes of these domains (Pathak
and London 2011).
It is not yet known to what extent this also holds for
SMA. However, there is evidence that suggests that SMA
may be less perturbing than detergent. It has been classified
as an extraordinarily mild solubilizing agent (Vargas et al.
2015; Cuevas Arenas et al. 2016) having a very low free
energy cost for solubilization of lipids from membranes
into SMALPs. This is reflected by the native-like bilayer
organization of the solubilized lipids (Jamshad et al. 2014).
The results in the present study furthermore indicate that
SMA is fully promiscuous in fluid bilayers, which suggests
that SMA does not significantly perturb membrane
homogeneity. Together, these results suggest that SMA could
serve as an alternative for the isolation of highly ordered
membrane domains that may have advantages over
conventional methods using cold detergent solutions. However,
whether indeed and to what extent SRMs isolated from
biological membranes are superior to DRMs remains to be
assessed.
Finally, an interesting novel possibility of this
application of SMA may lie in the size of ordered domains
existing in biological membranes. SMA may be capable of
solubilizing very small membrane nanodomains in a conserved
bilayer organization in case they are smaller than the
average size of the SMALPs. This may for the first time make
it possible to solubilize and characterize such small ordered
domains directly from native membranes at physiological
temperatures.
In this study, we show that in fluid membranes SMA does
not exhibit a preference for solubilization of specific lipids,
which supports the validity of studying preferential lipid–
protein interactions in SMA-bounded nanodiscs derived
from either model or biological membranes. In
phase-separated membranes, SMA has a strong preference for
solubilization of the fluid phase, with the potential application
of isolating ordered domains from biological membranes
by exploiting their SMA resistance. Our initial data suggest
that the use of SMA for these approaches may be an
alternative to cold detergent solutions, which are commonly
used for this purpose.
Acknowledgements This work was supported financially by NWO
Chemical Sciences, ECHO grant (no. 711-013-005) (J.J.D.P) and by
the Foundation for Fundamental Research on Matter (FOM),
program nos. 126 (S.S.) and 127 (A.I., V.S.) as well as by the Seventh
Framework Program of the European Union (Initial Training Network
“ManiFold,” grant 317371) (J.M.D.). We thank M. R. Hogeboom,
J. J. Rietveld, R. M. Hopman and H. Keser for optimizing the lipid
solubilization parameters. This work used the platforms of the
Grenoble Instruct centre (ISBG;UMS 3518 CNRS-CEA-UJF-EMBL)
with support from FRISBI (ANR-10-INSB-05-02) and GRAL
(ANR10-LABX-49-01) within the Grenoble partnership for structural
biology (PSB). We are very grateful to Dr. C. Moriscot from the Electron
Microscopy platform of the Integrated Structural Biology of Grenoble
(ISBG, UMI3265) for performing the TEM imaging and to B. W. M.
Kuipers from the University of Utrecht for advice and support in DLS
analysis.
Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a
link to the Creative Commons license, and indicate if changes were
made.
Bell AJ , Frankel LK , Bricker TM ( 2015 ) High yield non-detergent isolation of photosystem I-light-harvesting chlorophyll II membranes from spinach thylakoids . J Biol Chem 290 ( 30 ): 18429 - 18437 . doi:10.1074/jbc.m115.663872
Bligh EG , Dyer WJ ( 1959 ) A rapid method of total lipid extraction and purification . Can J Biochem Physiol 37 ( 8 ): 911 - 917
Brown DA , Rose JK ( 1992 ) Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface . Cell 68 ( 3 ): 533 - 544 . doi:10.1016/ 0092 -8674(92) 90189 - J
Cerneus DP , Ueffing E , Posthuma G , Strous GJ , van der Ende A ( 1993 ) Detergent insolubility of alkaline phosphatase during biosynthetic transport and endocytosis . Role of cholesterol. J Biol Chem 268 ( 5 ): 3150 - 3155
Cuevas Arenas R , Klingler J , Vargas C , Keller S ( 2016 ) Influence of lipid bilayer properties on nanodisc formation mediated by styrene/ maleic acid copolymers . Nanoscale . doi:10.1039/C6NR02089E
de Almeida RFM , Fedorov A , Prieto M ( 2003 ) Sphingomyelin/phosphatidylcholine/cholesterol phase diagram: boundaries and composition of lipid rafts . Biophys J 85 ( 4 ): 2406 - 2416 . doi:10.1016/ S0006-3495(03)74664- 5
de Smet CH , Vittone E , Scherer M , Houweling M , Liebisch G , Brouwers JF , de Kroon AIPM ( 2012 ) The yeast acyltransferase Sct1p regulates fatty acid desaturation by competing with the desaturase Ole1p . Mol Biol Cell 23 ( 7 ): 1146 - 1156 . doi:10.1091/mbc. e11- 07 - 0624
Dörr JM , Koorengevel MC , Schäfer M , Prokofyev AV , Scheidelaar S , van der Cruijsen EAW , Dafforn TR , Baldus M , Killian JA ( 2014 ) Detergent-free isolation, characterization, and functional reconstitution of a tetrameric K+ channel: the power of native nanodiscs . Proc Natl Acad Sci USA 111 ( 52 ): 18607 - 18612
Dörr JM , Scheidelaar S , Koorengevel MC , Dominguez JJ , Schäfer M , van Walree CA , Killian JA ( 2016 ) The styrene-maleic acid copolymer: a versatile tool in membrane research . Eur Biophys J 45 ( 1 ): 3 - 21 . doi:10.1007/s00249- 015 - 1093 -y
El Kirat K , Morandat S ( 2007 ) Cholesterol modulation of membrane resistance to Triton X-100 explored by atomic force microscopy . Biochim Biophys Acta Biomembr 1768 ( 9 ): 2300 - 2309 . doi:10.1016/j.bbamem. 2007 .05.006
Gulati S , Jamshad M , Knowles TJ , Morrison KA , Downing R , Cant N , Collins R , Koenderink JB , Ford RC , Overduin M , Kerr ID , Dafforn TR , Rothnie AJ ( 2014 ) Detergent-free purification of ABC (ATP-binding-cassette) transporters . Biochem J 461 ( 2 ): 269 - 278 . doi:10.1042/BJ20131477
Hanado K , Nishijima M , Akamatsu Y , Pagano RE ( 1995 ) Both sphingolipids and cholesterol participate in the detergent insolubility of alkaline phosphatase, a glycosylphosphatidylinositolanchored protein, in mammalian membranes . J Biol Chem 270 ( 11 ): 6254 - 6260 . doi:10.1074/jbc.270.11.6254
Heerklotz H ( 2002 ) Triton promotes domain formation in lipid raft mixtures . Biophys J 83 ( 5 ): 2693 - 2701 . doi:10.1016/ s0006-3495(02)75278- 8
Ipsen JH , Karlstrom G , Mouritsen OG , Wennerstrom H , Zuckermann MJ ( 1987 ) Phase equilibria in the phosphatidylcholine-cholesterol system . Biochim Biophys Acta 905 ( 1 ): 162 - 172
Jamshad M , Grimard V , Idini I , Knowles TJ , Dowle MR , Schofield N , Sridhar P , Lin Y , Finka R , Wheatley M et al ( 2014 ) Structural analysis of a nanoparticle containing a lipid bilayer used for detergent-free extraction of membrane proteins . Nano Res . 8 ( 3 ): 774 - 789 . doi:10.1007/s12274- 014 - 0560 -6
Jamshad M , Charlton J , Lin Y-P , Routledge SJ , Bawa Z , Knowles TJ , Overduin M , Dekker N , Dafforn TR , Bill RM et al ( 2015 ) G-protein coupled receptor solubilization and purification for biophysical analysis and functional studies, in the total absence of detergent . Biosci Rep 35 ( 2 ): 1 - 10 . doi:10.1042/bsr20140171
Klymchenko AS , Kreder R ( 2014 ) Fluorescent probes for lipid rafts: from model membranes to living cells . Chem Biol 21 ( 1 ): 97 - 113 . doi:10.1016/j.chembiol. 2013 .11.009
Lewis RN , Mak N , McElhaney RN ( 1987 ) A differential scanning calorimetric study of the thermotropic phase behavior of model membranes composed of phosphatidylcholines containing linear saturated fatty acyl chains . Biochemistry 26 ( 19 ): 6118 - 6126
Lichtenberg D , Goñi FM , Heerklotz H ( 2005 ) Detergent-resistant membranes should not be identified with membrane rafts . Trends Biochem Sci 30 ( 8 ): 430 - 436 . doi:10.1016/j.tibs. 2005 .06.004
London E , Brown DA ( 2000 ) Insolubility of lipids in Triton X-100: physical origin and relationship to sphingolipid/cholesterol membrane domains (rafts) . Biochim Biophys Acta Biomembr 1508 ( 1-2 ): 182 - 195 . doi:10.1016/S0304-4157(00)00007- 1
Long AR , O'Brien CC , Malhotra K , Schwall CT , Albert AD , Watts A , Alder NN ( 2013 ) A detergent-free strategy for the reconstitution of active enzyme complexes from native biological membranes into nanoscale discs . BMC Biotechnol 13 :41. doi:10.1186/ 1472 - 6750 - 13 - 41
Marsh D ( 2013 ) Handbook of lipid bilayers, 2nd edn . CRC Press, Boca Raton
McMullen TP , Lewis RN , McElhaney RN ( 2004 ) Cholesterol-phospholipid interactions, the liquid-ordered phase and lipid rafts in model and biological membranes . Curr Opin Colloid Interface Sci 8 ( 6 ): 459 - 468 . doi:10.1016/j.cocis. 2004 .01.007
Nibu Y , Inoue T , Motoda I ( 1995 ) Effect of headgroup type on the miscibility of homologous phospholipids with different acyl chain lengths in hydrated bilayer . Biophys Chem 56 ( 3 ): 273 - 280
Orwick MC , Judge PJ , Procek J , Lindholm L , Graziadei A , Engel A , Gröbner G , Watts A ( 2012 ) Detergent-free formation and physicochemical characterization of nanosized lipid-polymer complexes: lipodisq . Angew Chem Int Ed Engl 51 ( 19 ): 4653 - 4657 . doi:10.1002/anie.201201355
Orwick-Rydmark M , Lovett JE , Graziadei A , Lindholm L , Hicks MR , Watts A ( 2012 ) Detergent-free incorporation of a seventransmembrane receptor protein into nanosized bilayer Lipodisq particles for functional and biophysical studies . Nano Lett 12 ( 9 ): 4687 - 4692 . doi:10.1021/nl3020395
Pathak P , London E ( 2011 ) Measurement of lipid nanodomain (raft) formation and size in sphingomyelin/POPC/cholesterol vesicles shows TX-100 and transmembrane helices increase domain size by coalescing preexisting nanodomains but do not induce domain formation . Biophys J 101 ( 10 ): 2417 - 2425 . doi:10.1016/j. bpj. 2011 .08.059
Patra SK , Alonso A , Arrondo JL , Goñi FM ( 1999 ) Liposomes containing sphingomyelin and cholesterol: detergent solubilisation and infrared spectroscopic studies . J Liposome Res 9 ( 2 ): 247 - 260 . doi:10.3109/08982109909024788
Prabudiansyah I , Kusters I , Caforio A , Driessen AJ ( 2015 ) Characterization of the annular lipid shell of the Sec translocon . Biochim Biophys Acta Biomembr 1848 ( 10 ): 2050 - 2056 . doi:10.1016/j. bbamem. 2015 .06.024
Rinia HA , Snel MM , van der Eerden JP , de Kruijff B ( 2001 ) Visualizing detergent resistant domains in model membranes with atomic force microscopy . FEBS Lett 501 ( 1 ): 92 - 96
Rouser G , Fleischer S , Yamamoto A ( 1970 ) Two dimensional thin layer chromatographic separation of polar lipids and determination of phospholipids by phosphorus analysis of spots . Lipids 5 ( 5 ): 494 - 496 . doi:10.1007/bf02531316
Sankaram MB , Thompson TE ( 1990 ) Interaction of cholesterol with various glycerophospholipids and sphingomyelin . Biochemistry 29 ( 47 ): 10670 - 10675
Scheidelaar S , Koorengevel MC , Pardo JD , Meeldijk JD , Breukink E , Killian JA ( 2015 ) Molecular model for the solubilization of membranes into nanodisks by styrene maleic acid copolymers . Biophys J 108 ( 2 ): 279 - 290 . doi:10.1016/j. bpj. 2014 .11.3464
Schroeder R , London E , Brown D ( 1994 ) Interactions between saturated acyl chains confer detergent resistance on lipids and glycosylphosphatidylinositol (GPI)-anchored proteins: GPI-anchored proteins in liposomes and cells show similar behavior . Proc Natl Acad Sci 91 ( 25 ): 12130 - 12134
Simons K , Ikonen E ( 1997 ) Functional rafts in cell membranes . Nature 387 ( 6633 ): 569 - 572 . doi:10.1038/42408
Sot J , Collado MI , Arrondo JLR , Alonso A , Goñi FM ( 2002 ) Triton X-100-resistant bilayers: effect of lipid composition and relevance to the raft phenomenon . Langmuir 18 ( 7 ): 2828 - 2835 . doi:10.1021/la011381c
Swainsbury DJK , Scheidelaar S , van Grondelle R , Killian JA , Jones MR ( 2014 ) Bacterial reaction centers purified with styrene maleic acid copolymer retain native membrane functional properties and display enhanced stability . Angew Chem Int Ed Engl 53 ( 44 ): 11803 - 11807 . doi:10.1002/anie.201406412
Vargas C , Arenas RC , Frotscher E , Keller S ( 2015 ) Nanoparticle selfassembly in mixtures of phospholipids with styrene/maleic acid copolymers or fluorinated surfactants . Nanoscale 7 ( 48 ): 20685 - 20696 . doi:10.1039/c5nr06353a
Veatch SL , Keller SL ( 2003 ) Separation of liquid phases in giant vesicles of ternary mixtures of phospholipids and cholesterol . Biophys J 85 ( 5 ): 3074 - 3083 . doi:10.1016/ s0006-3495(03)74726- 2
Veiga MP , Arrondo JLR , Goñi FM , Alonso A , Marsh D ( 2001 ) Interaction of cholesterol with sphingomyelin in mixed membranes containing phosphatidylcholine, studied by spin-label esr and ir spectroscopies. A possible stabilization of gel-phase sphingolipid domains by cholesterol . Biochemistry 40 ( 8 ): 261 - 2622 . doi:10.1021/bi0019803
Wan C-PL , Chiu MH , Wu X , Lee SK , Prenner EJ , Weers PMM ( 2011 ) Apolipoprotein-induced conversion of phosphatidylcholine bilayer vesicles into nanodisks . Biochim Biophys Acta Biomembr 1808 ( 3 ): 606 - 613 . doi:10.1016/j. bbamem. 2010 .11.020
Zhang L , Song J , Cavigiolio G , Ishida BY , Zhang S , Kane JP , Weisgraber KH , Oda MN , Rye K-A , Pownall HJ , Ren G ( 2011 ) Morphology and structure of lipoproteins revealed by an optimized negative-staining protocol of electron microscopy . J Lipid Res 52 ( 1 ): 175 - 184 . doi:10.1194/jlr.D010959
Zhang R , Sahu ID , Liu L , Osatuke A , Comer RG , Dabney-Smith C , Lorigan GA ( 2015 ) Characterizing the structure of lipodisq nanoparticles for membrane protein spectroscopic studies . Biochim Biophys Acta Biomembr 1848 ( 1 ): 329 - 333 . doi:10.1016/j. bbamem. 2014 .05.008