Monoolein Lipid Phases as Incorporation and Enrichment
Materials for Membrane Protein Crystallization
Ellen Wallace1, David Dranow1, Philip D. Laible2, Jeff Christensen1, Peter Nollert1*
1 Emerald BioStructures, Bainbridge Island, Washington, United States of America, 2 Biosciences Division, Argonne National Laboratory, Argonne, Illinois, United States of
America
Abstract
The crystallization of membrane proteins in amphiphile-rich materials such as lipidic cubic phases is an established
methodology in many structural biology laboratories. The standard procedure employed with this methodology requires
the generation of a highly viscous lipidic material by mixing lipid, for instance monoolein, with a solution of the detergent
solubilized membrane protein. This preparation is often carried out with specialized mixing tools that allow handling of the
highly viscous materials while minimizing dead volume to save precious membrane protein sample. The processes that
occur during the initial mixing of the lipid with the membrane protein are not well understood. Here we show that the
formation of the lipidic phases and the incorporation of the membrane protein into such materials can be separated
experimentally. Specifically, we have investigated the effect of different initial monoolein-based lipid phase states on the
crystallization behavior of the colored photosynthetic reaction center from Rhodobacter sphaeroides. We find that the
detergent solubilized photosynthetic reaction center spontaneously inserts into and concentrates in the lipid matrix
without any mixing, and that the initial lipid material phase state is irrelevant for productive crystallization. A substantial in-
situ enrichment of the membrane protein to concentration levels that are otherwise unobtainable occurs in a thin layer on
the surface of the lipidic material. These results have important practical applications and hence we suggest a simplified
protocol for membrane protein crystallization within amphiphile rich materials, eliminating any specialized mixing tools to
prepare crystallization experiments within lipidic cubic phases. Furthermore, by virtue of sampling a membrane protein
concentration gradient within a single crystallization experiment, this crystallization technique is more robust and increases
the efficiency of identifying productive crystallization parameters. Finally, we provide a model that explains the
incorporation of the membrane protein from solution into the lipid phase via a portal lamellar phase.
Citation: Wallace E, Dranow D, Laible PD, Christensen J, Nollert P (2011) Monoolein Lipid Phases as Incorporation and Enrichment Materials for Membrane
Protein Crystallization. PLoS ONE 6(8): e24488. doi:10.1371/journal.pone.0024488
Editor: Petri Kursula, University of Oulu, Germany
Received March 27, 2011; Accepted August 11, 2011; Published August 31, 2011
Copyright:  2011 Wallace et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was funded by the NIH Roadmap grant P01 GM075913. The funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: EW, DD, JC and PN are employed by Emerald BioStructures, Inc. that markets, together with its sister company Emerald BioSystems, Inc.
crystallization tools and services. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials.
* E-mail: pnollert@embios.com
Introduction
Lipidic cubic phases and related amphiphile-rich materials have
served as matrices for growing a variety of membrane protein
crystals [1], the latter of which were used in determining X-ray
crystallographic structures of several high-impact target proteins
such as G-protein coupled receptors [2,3,4,5]. The procedures and
tools employed to grow such membrane protein crystals have been
refined over the past 15 years (Figure 1) and are used in many
membrane protein crystallization laboratories [6,7]. Initially,
crystallizations were carried out as batch experiments in small test
tubes with ca. 10 mL total setup volume [8,9]. Soon after, a
procedure employing positive displacement devices for the
preparation of crystallization experiments in dedicated crystalliza-
tion plates was introduced [10], later reproduced [11,12], and
refined with the goal to further reduce setup volumes and increase
expediency [13]. Most of these technological developments aimed at
improving the tools that manipulate small volumes of the highly
viscous LCP (lipidic cubic phase) that is obtained when monoolein is
mixed with a membrane protein solution [14]. Attempts have been
made to avoid the requirement for dealing with the highly viscous
LCP, such as devising protocols for crystallization within sponge
phases [15], which are runny liquids that can be handled with
standard laboratory pipettors, and were instrumental to produce
crystals and a 1.86 A˚ crystallographic structure of the reaction
center from Blastochloris viridis [15]. A drawback of this method is the
requirement to add often undesired sponge phase-inducing reagents
to crystallization experiments [16]. Recently, it has been reported
that crystals of Photosynthetic Reaction Center from Rhodobacter
sphaeroides and Blastochloris viridis can be obtained using a microfluidic
device wherein the protein solution is mixed with an already
established lipidic cubic phase material. This method was named
PLI, post lipidic cubic phase formation incorporation [17].
Here we test different lipid phases that monoolein spontane-
ously forms with water and explore their utility in providing a
matrix for membrane protein crystallization experiments. We aim
to adapt the PLI preparation methodology to techniques that are
compatible with standard laboratory liquid dispensation tools and
practices (Figure 1). We also investigate the early stages of this new
crystallization regime, namely the incorporation of the membrane
protein RC (Photosynthetic Reaction Center from Rhodobacter
sphaeroides) into a lipidic phase prior to crystallization.
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Results
1 RC Crystallization according to the PLI methodology
We have scaled up and adapted the PLI membrane protein
crystallization methodology to be compatible with standard
pipetting tools (Figure 1), as opposed to microfluidic devices
[17], and applied it to the crystallization of RC. In fact, any
monoolein lipid phase can be employed (Figure S1) to crystallize
RC solubilized from LDAO (Lauryldimethylamine-oxide) which is
first added to the lipid and then combined with precipitation
reagents. In our experiments RC crystals grew and their X-ray
diffraction limit was similar, regardless of the initial monoolein
hydration level as long as monoolein was present (Figure 2,
Figure 3, see also supplemental material, specifically Figure S2).
2 Optimized RC/monoolein pre-incubation time
In order to devise a simplified PLI membrane protein
crystallization protocol [17], we investigated the effect of the
duration of the RC solution exposure to the lipid phase (Figure 4).
We found that PLI setups yielded crystals for those experiments
where the RC sample was incubated with monoolein for a time
period of 2 hours to 2 days prior to addition of the precipitation
reagent. The optimal incubation time was about half a day,
conveniently carried out overnight. We noticed that the overall
success of pre-incubation of RC with monoolein as compared to
mechanical mixing of the protein solution with monoolein is
substantially higher.
3 RC enrichment
In order to better examine the first step in the PLI process,
incorporation of RC into LCP, we prepared thin sandwich setups
similar to those described by Cherezov et al. [18] to enhance the
optical inspection path through the setup, reduce aberrations and
improve the interpretation of generated images. We took
advantage of the chromophores within RC [19] to optically track
the diffusion and concentration of RC in microscopy images by
virtue of their red/purple color. The spontaneous enrichment of
RC at the interface of dispensed LCP and detergent solubilized
RC solution is evident from the darkening of the ca. 0.1 millimeter
thick rim section around the LCP material (Figure 5) after
exposure of the sandwiched LCP bolus to RC containing solution.
Within minutes of initial exposure the RC color saturation and
hence the RC concentration increases at this rim and reaches a
peak after ca. 5 hours, corresponding to an approximate 3.3-fold
enrichment within the lipid material at this location, as judged by
the increase in color saturation. We wished to determine an
Figure 1. Brief diagrammatic history of the development of LCP-based crystallization techniques (,15 years). A: Batch experiments
carried out in micro test tubes [8]. Here, solid monoolein is combined with protein solution and precipitating reagents (1) and mixing is by 180-
degree rotation of tube between centrifugation cycles (2,3). Each trial requires several microliters of protein and a minimum of 2 hours of preparation
time (with typically a maximum of 24 simultaneous experiments); B: Syringe-based crystallization experiments where proteo-LCP is first prepared and
then dispensed directly into precipitating reagents in crystallization trays, involving a four-step process: (I) Proteo-LCP is initially formed by coupling
two syringes (I; one filled with 60% monoolein and the other with 40% protein solution) and by mixing of the two components with repetitive cycling
of the entire combined volume from one barrel to the other. (II) Precipitant solutions fill the wells of a crystallization tray (4), a single well also shown
(5). (III) Proteo-LCP is dispensed to each microwell with a semi-automatic ratchet dispenser (3, 6) after the material is transferred into a microsyringe
(2). (IV) The experiments are sealed with clear transparent tape (8) and stored (9). The Proteo-LCP is stable in an excess of overlaying liquid (7). Crystals
appear only within the lipid matrix (10). Proteo-LCP is dispensed into the precipitating reagents to avoid detrimental dehydration. A kit (Cubic LCP kit,
Emerald BioSystems, Bainbridge Island, WA USA) and robotic versions of this dispensation technique [13] are available. Each experiment utilizes
,200 nL of proteo-LCP – minimizing protein requirements and allowing for hundreds of precipitants to be screened simultaneously; C. PLI
approaches, as adapted from [17], dispense fluid lipid materials into microwells using airtight syringes (1) prior to the addition of a solution of
membrane protein by conventional pipetting (2). After a delay that allows the membrane proteins to integrate into the lipidic material (3),
precipitating reagents are added (4) and the wells are sealed and stored (5). Here, the precipitating reagent dilutes the remaining unincorporated
membrane protein solution. Crystals, again, only appear within the lipid matrix. PLI approaches also minimize protein requirements and are amenable
to high-throughput approaches utilizing automated liquid handlers.
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independent estimation of this transient enrichment based on the
difference in volume occupied by RC at the start and end of the
experiment. Initially, the RC occupied an area of 14.47 mm2,
calculated by the difference between the total area of the well
(19.64 mm2) and the initial LCP bolus (5.17 mm2). The original
area was compared to the final occupied area, which we calculated
to be 1.98 mm2, based on the difference between the area defined
by the outer ring of the bolus and the inner ring of the clear area of
the bolus. This gives a fold increase of,7.3. For these calculations
we chose to ignore the volume, as the sandwich plate creates a
consistent height in all objects contained within. We suspect that
these differences in these RC enrichment estimates are due to the
inexact correlation of the image-based volume and color
saturation with actual RC concentrations. Further, more quanti-
Figure 2. Images of the steps involved in conducting a PLI crystallization experiment with RCs using either neat, dry monoolein
(image sequence A) or preformed LCP (40% water 60% monoolein; image sequence B). The process begins by adding 0.2 ml of the lipid or
lipid mixture to the empty wells (1), of ca. 2 mm diameter, resulting in the second image in the series (2). Following sequential additions of RC
solution (3; 0.4 ml) and precipitating solution (4, 2 ml in drop and 80 ml in reservoir), crystals were observed after 2 days (5). Magnified images of RC
crystals are shown on the right. In these specific experiments, RCs were incubated with lipids for 4 hours prior to the addition of precipitating
solution (1 M HEPES, pH 7.5, 1.15 M ammonium sulfate, Jeffamine M-600, 12% v/v). The top table tallies the components that are present at the time
the images were taken.
doi:10.1371/journal.pone.0024488.g002
Figure 3. Yields of successful RC crystallization trials from independent PLI experiments where the initial monoolein hydration
state and concentration of the precipitating agent, Jeffamine, were independently varied. The data represent results from highly
replicated experiments and where RCs were allowed to incubate and integrate into lipid mixtures overnight prior to addition of precipitating
solutions (which included 1 M HEPES/NaOH, pH 7.5 and 1.15 M ammonium sulfate in addition to Jeffamine, as indicated). Exemplary images of
crystals observed 7 days after set up are shown for one particular replicate, each ca. 1006100 mm sections. Where images are absent, crystals were of
poor quality or not observed for this particular trial. Crystal yield [positive/attempted] refers to the number of trials in which crystals were observed
(positive) relative to the number of trials in which lipid, protein and crystallant all made contact (attempted). Diffraction limits were determined using
an in-house X-ray source.
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tative studies are required to accurately assess the transient
concentration of the membrane protein close to the solution
exposed LCP.
The interface displays phenomena that are related to a number
of processes, including the insertion of RC into the lipid phase, the
expansion of the lipid phase, and possibly, additional phase
transition and optical effects that are due to refractive index
differences and light scattering from surfaces and at interfaces.
Our interpretation as presented above assumes that the measured
color saturation changes are dominated by local concentrations of
RC. While the traces in Figure 5D contain information regarding
the kinetics of partitioning and diffusion, additional experimenta-
tion, with apparati designed for increased control of parameters, is
required in order to quantitatively assess coefficients and rate
constants for these processes.
Nevertheless, the images clearly show that the RC enrichment
at the rim section is of transient nature and involves the formation
of an RC gradient towards the center of the LCP. At this rim, the
initial increase in saturation is followed by a decline (Figure 5C,
D). The final state of complete RC depletion in the solution and
equilibrated RC throughout the bulk of the LCP is not reached
during the timescale of a crystallization experiment, thus
presenting an RC concentration gradient at the time of
precipitation reagent addition. The ca. 0.1 mm thick rim section
consists of several distinct, ca. 20–50 micrometer thick zones
(Figure 5C), each exhibiting unique RC enrichment kinetics
(Figure 5D). The comprehensive interpretation of the development
of these zones is exacerbated by the dynamic nature of the rim,
possibly caused by several simultaneous processes occurring during
the course of the experiment. For instance, we observe an overall
34% hydration-triggered LCP expansion, the formation of distinct
zones, and diffusion of RC. Importantly, the images of the rim
section and their time dependent changes in color saturation
clearly demonstrate that, within the timescale of a typical RC
crystallization experiment, RC enriches to different levels within
sections of the outer layers of the monoolein material (Figure 5C
and D). During the course of incubation the reservoir of RC
depletes in the aqueous solution, and the RC shows some
equilibration within the bulk of the LCP by diffusion. The
timescale of the enrichment is compatible with the diffusion of
membrane proteins within LCP [20].
While the bulk monoolein cubic phase remained transparent
and non-birefringent, the solution-exposed surface appeared shiny
under microscopic inspection using crossed polarizers (not shown).
We speculate that a thin section of the lipid material forms a portal
lamellar phase at the solution exposed surface (Figure 6), allowing
detergent and RC molecules to enter a bilayer structure that is
connected to the curved lipid bilayer system within the LCP, a
mechanism that is similar to the crystal growth hypothesis brought
forward initially by [21] and later verified [22].
Discussion
Our main result is that RC can be crystallized without
mechanical mixing in monoolein-based matrices, regardless of
the initial lipid phase state employed during pre-incubation. The
presence of lipid bulk material is required for crystallization
though, since RC crystals did grow only in lipid containing
experiments. Hence, a specific interaction of the RC with the lipid
phase is required for crystallization. We have shown that the
interaction of RC with the LCP encompasses (i) a substantial
transient RC enrichment at the LCP solution interface, and thus,
(ii) the formation of a RC concentration gradient within the LCP
during a timescale that is relevant for crystallization to occur.
These phenomena are desired features in membrane protein
crystallization experiments since the concentration effect increases
the particle density, hence assuring supersaturation conditions
within the crystallization experiment. Furthermore, the formation
of a membrane protein concentration gradient within the matrix
lipid constitutes an effective, continuous sampling of many
different protein concentrations within a single crystallization
experiment [23,24]. The combination of these two features in the
PLI-crystallization method [17] nicely explains the increased
robustness and higher crystallization hit rate of ca. 25% as
compared to standard LCP crystallization experiments, the latter
of which require complicated pre-mixing of lipidic cubic phase
materials [25].
Finally, the spontaneous insertion of the membrane protein
from the detergent phase into the bilayer organization of the LCP
is compatible with the current understanding of the mechanistic
aspects (Figure 6) of crystallization of membrane proteins within
lipidic cubic phases [21,26]. We infer that the exposure of LCP to
the RC solution initiates hydration of the LCP and fast
partitioning of the detergent into the LCP. Detergents such as
LDAO have been shown to form lamellar structures in ternary
mixtures of monoolein, water and detergent [27]. While not
observed directly, we assume that in our experiments LDAO
initially enriches in an outer layer of the LCP as it partitions into
the LCP, similar to RC as demonstrated in Figure 5. While the
LDAO concentration in the bulk solution is not sufficient to
convert the entire LCP into a lamellar phase, it is conceivable that
its transient enrichment in the outer rim suffices to form
membranous structures with low curvature. Such portal lamellar
structures could form the entrance points for RC to fuse with and
become part of the bulk LCP via diffusion (Figure 6).
The standard micro LCP crystallization method [10] (Figure 1B)
is carried out by mixing the detergent solubilized protein solution
with dry lipid, yielding an LCP with incorporated membrane
protein within less than a minute. This substantially faster
membrane protein incorporation into LCP using the syringe-
based mixer method is presumably caused by the employed
turbulent mixing regime, forming large interaction surfaces
between lipid and solution, making the membrane protein
incorporation process very efficient.
Figure 4. Effect of the length of RC/monoolein pre-incubation
periods on the yield of productive crystallization experiments.
Crystal yield is given as the number of successful experiments out of a
total of 12 conducted for each pre-incubation period. Experiments
utilized neat, dry lipid dispensed in molten form at 37uC. Crystallization
success was judged 4 days after precipitant addition.
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Taken together, the reported findings have several practical
consequences: (i) Membrane protein crystallization with pre-
dispensed lipidic phases are greatly simplified (Figure 1) because
the handling of the lipid material and the membrane protein
solution are separated processes that can be carried out
independently of each other and with suitable dispensation tools.
For instance, the dispensation of the highly viscous lipidic cubic
phase with positive displacement syringes can be replaced by the
dispensation of relatively low viscosity molten monoolein lipid.
This enables the testing of very small quantities of precious
membrane protein samples as these samples are not required to be
mixed with coupled syringe devices (250 ml) bearing about a 5 ml
dead volume, [10,11,12]. Hence, crystallization plates with pre-
dispensed lipids can be prepared in advance and made
commercially available (i.e. NeXtalCubicPhase uplate; Qiagen,
Hilden, Germany). Indeed, crystals of Sensory Rhodopsin II
(H.Salinarum) and that of an unidentified G-protein coupled
receptor protein have been obtained using this approach (personal
communication, Frank Schaefer Qiagen, http://www.qiagen.
com/literature/render.aspx?id = 104833). Within a relatively
short period of time, productive crystal growth of four different
membrane proteins has been reported with the PLI approach.
Within a relatively short period of time, productive crystal growth
of four different membrane proteins has been reported with the
PLI approach. While we think this bodes well for the applicability
of this protocol to membrane proteins in general, a careful
comparative analysis of productive crystallizations is required to
fully appreciate its utility. (ii) The initial lipid hydration extent, the
period of incubation with the membrane protein solution and the
crystallization setup geometry, specifically the size of the exposed
lipid material surface area, add further crystallization optimization
parameters to potentially improve the quality of membrane
protein crystals. (iii) Similar to gel-based gradient crystallization
methods [23,24], the sampling of many different membrane
protein concentrations in a membrane protein concentration
gradient within a single setup enhances the efficiency and
Figure 5. Tracking RC migration into the lipid matrix reveals the existence of concentration gradients. Here, the process of
incorporation of RCs from solution into the bulk LCP is shown in a two-dimensional sandwich arrangement in the absence of precipitating solution. A:
Initial image at ,20 seconds post addition of 2.5 ml of RC solution (20 mg/ml) to a 0.4 ml bolus of LCP prepared with 44% water and 56% monoolein.
Air bubbles from the RC solution preferentially adhere to the LCP (center) and transparent adhesive seal. B: Additional image after 16 hours of
incubation. Here, RCs are depleted from the aqueous solution and enriched at the LCP/solution interface, and the central LCP area is devoid of RCs.
RC concentrations may approach 146 mg/ml in the enrichment zone (7.36enrichment factor) if the entire RC addition is localized to the area that the
colored RC occupied at the interface. The observed 34% increase in area observed for the LCP matches that expected to occur as monoolein
hydration increases from 44% to 58% (the latter is the maximum hydration of LCP at 16uC). Scale bar in A and B is 2 mm. C: Magnified image (scale
bar = 0.2 mm) of the six enriched zones that were monitored closely. RC concentrations were tracked in the bulk solution (Zone 1), the LCP/RC
solution interface (Zone 2), and regions within the LCP at increasing distance from the LCP/RC solution interface (Zones 3, 4, 5, and 6). Color
enhancement in Zone 4 is maximal at 16 hours and represents 3.3 times that of the initial color intensity of Zone 1 at the start of the experiments.
Thus, there is an approximate 3-fold enrichment of RC concentration within the LCP in this zone. D: Quantitation of RC concentration using color
saturation values of images, like those in A and B. Here, it is most evident that the concentration of RC in region 1 rapidly decreases and stabilizes at a
minimum after ,1 hour. The concentration of RC in the interior of the bulk LCP (region 6) increases only slightly throughout the experiment,
indicating slow RC migration/equilibrium throughout the LCP. Zones 2 and 3 are initially part of the RC solution. These regions become enriched in
RCs after 4 and 10 hours of incubation, respectively, as RCs migrate back to the aqueous liquid from the most rapidly- and highly-enriched Zones 4
and 5. Thus, after initially migrating directionally into the LCP and concentrating in Zone 5, the RCs subsequently migrate/diffuse freely in both
directions (not only further to the interior of the LCP, zone 6, but also back towards the bulk aqueous solution). Zones 4 and 5 experience the largest
increases in color saturation, with Zone 5 showing a distinct maximum at 5 hours, followed by a steady decline, possibly to the benefit of Zone 6.
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robustness of the crystallization experiment. Thus in primary
screening experiments more parameters are sampled, enhancing
the success rate to identify productive membrane protein
crystallization conditions. (iv) Since the membrane proteins
spontaneously concentrate at the lipid material surface, samples
that would typically be considered unfit for crystallization
experiments owing to their low protein content may be subjected
to PLI crystallization trials. Indeed, we have demonstrated (data
not shown) that RC crystals can be grown from diluted RC
solutions (i.e. 2.5 mg/ml) using the PLI method. This is a
significant advantage over traditional crystallization methods
because the generation of membrane protein samples with high
protein concentrations, typically exceeding 10 mg/ml, is often the
main experimental barrier for membrane protein crystallization
trials. The observed concentration factors of 3.3 to 7.3 relax this
requirement substantially. Furthermore, this concentration effect
may be used to enrich membrane proteins for purposes other than
for crystallization, for instance for functional assays or storage. (v)
Compared to mixing of LCP in coupled syringes where high shear
stress is exerted on the lipid matrix and the membrane protein, the
incubation of the membrane protein solution with portions of pre-
dispensed lipid provide gentler reconstitution conditions, the latter
of which may aid the application of labile membrane proteins to
such crystallization trials. Hence it extends the crystallization
optimization repertoire for those cases where mixing with
monoolein destabilizes or renders the protein uncrystallizable
[12,28]. On the other hand, faster incorporation of the membrane
protein, brought about by mechanical mixing, may be a gentler
procedure for proteins that are less stable in the detergent phase
than in the LCP at room temperature.
Aside from these practical aspects, the reconstitution of
membrane proteins from a mixed membrane protein detergent
complex and detergent micelle phase into a lipid bilayer system is
of fundamental interest to membrane protein research. While the
details of the membrane protein incorporation processes into the
bilayer structure of an LCP remain poorly understood, we
hypothesize that the partitioning of detergent into the LCP
promotes the formation of lamellar structures that aid the insertion
of detergent solubilized membranes into the bilayer structure of
the LCP (Figure 6). We note that this new experimental format
provides a simple system that allows dissecting the processes
involved in membrane protein reconstitution. Unlike the homog-
enous reconstitutions in solution, this PLI system provides a
heterogeneous experimental system with spacial fixation of the
lipid bulk allowing for detailed investigation of processes that ensue
during the incorporation of membrane proteins into membranes.
Figure 6. Illustration depicting how membrane proteins might incorporate into LCP in a PLI experiment. Solubilized membrane
proteins (yellow and gray) are associated with native lipids (orange and gray) and are complexed into detergent micelles, the latter of which are in
equilibrium with free detergent molecules (blue and gray). The relatively fast exchange of free detergent molecules with micellar structures allows for
facile partitioning of detergent into the bilayer structure of the bulk LCP. The indicated ki are time constants describing incorporation (k1),
solubilization (k2) and clearance from the interface (k3). According to this model, productive incorporation from the micellar phase occurs if k1.k2
and interfacial concentration occurs only if diffusion is slow as compared to the incorporation step (k1.k3).Detergents have been shown to
dramatically decrease the curvature of monoolein-based LCP [30], likely resulting in altered mesophase arragements of protruding bilayers consisting
of monoolein (green and gray) and detergent molecules (blue and gray; in our case the detergent is LDAO) that serve as portals for membrane
protein incorporation. These structures could promote the integration of membrane proteins into the curved, cubic, bulk material since they are
extensions from that phase. Once assimilated, membrane proteins diffuse readily in LCP, with rate constants that are similar to those in planar
bilayers, and are free to form nuclei and/or join growing crystals [20].
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Materials and Methods
1 Preparation of RC samples
Samples of R. sphaeroides RCs, solubilized and purified using the
detergent LDAO were prepared as described in [25]. RC
concentration was 20 mg/ml and solubilized in 10 mM Tris,
pH 7.8, 0.05% (w/v) LDAO, 280 mM NaCl. Small aliquots of
RC were shock frozen in liquid Nitrogen, stored at 280uC and
thawed quickly prior to use [29].
2 Preparation and Characterization of Monoolein-based
lipid phases
Monoolein phases were prepared by melting monoolein (Nu-
Check, Elysian, Minnesota, USA) and mixing with water using a
syringe-based apparatus as described [10]. In short, molten
monoolein was filled into one RN-type 250 micro liter syringe
(Hamilton, Reno, NV) and water was filled into a second syringe.
The syringes were joined with a coupler (Emerald BioSystems,
Bainbridge Island, WA) and a homogenous mixture was created
by pumping the content of one syringe into the other, with more
than 50 repeats. The final volume typically consisted of ca. 50–
100 ml lipidic material; for example, to prepare a 30% v/w water/
monoolein mixture one would combine 30 ml water with 40 mg
monoolein. The optical properties of the obtained materials were
assessed with and without crossed linear polarization filters
(Figure 1). Only mixtures with 30% v/w, 40% v/w and 100%
v/w water content in monoolein were transparent and non-
birefringent, and all remaining phases were turbid or birefringent,
as expected from isotropic lipid materials [30,31,32]. The
rheological properties were crudely characterized by measuring
the force required to pump the lipid material through the coupler
from one syringe into another syringe. This was done by reading
the weight measured when the syringe plunger of the assembly was
placed onto a balance and the coupled syringe contraption was
operated by pushing the plunger against the balance. Weight
readings were taken when the resistance to push the plunger of the
lipid filled syringe contraption was overcome. All lipid materials
passed through the same syringe and coupler for all measure-
ments. The highest resistance, found in the 30% v/w water/
monoolein mixture, was set to 100%. The average standard
deviation of such viscosity measurements was 8% (N=30). Only
lipid samples prepared with monoolein with 30% v/w and 40% v/
w hydration displayed the hallmark properties of lipidic cubic
phases: transparency, non-birefringence and high viscosity. The
assignment of the obtained materials to their respective lipid phase
type (Figure S1) is in perfect agreement with published monoolein
phase diagrams [30,31,32]. While the monoolein phase diagram
[33] shows stable LCP only for temperatures above 18uC, the
materials we obtained showed all the hallmark properties of lipidic
cubic phases. We speculate that such cubic phases form at slightly
lower temperatures due to the presence of LDAO, sodium
chloride and RC.
3 RC crystallization trials
RC crystallization experiments were carried out by adapting the
crystallization recipes as previously described [1,25,34]. In short,
ca. 0.2 ml of lipidic material were placed into a drop well of a
crystallization plate at 16uC (Clover Jr. plate, Emerald BioSystems,
Bainbridge Island, WA). To this, 0.4 ml RC sample were added to
the lipid and incubated for various times. Following incubation,
80 ml precipitant solution were added to the reservoir, and from
this 2 ml were transferred to the drop well, the plate sealed with
transparent tape, and the plate incubated at 16uC. In the case of
0% protein/100% monoolein, the monoolein was melted (37uC)
in order to aspirate it into a pre-warmed (37uC) ratchet dispenser,
allowing repeated dispensation of supercooled monoolein in
portions of 0.2 ml. All other phases were prepared via the syringe
coupling apparatus (Figure 1B). Crystallization experiments were
wrapped in foil to minimize exposure to light, and stored at 16uC.
Experiments were inspected 48–72 hours after set up with a Leica
MZ12.5 microscope. RC crystallization conditions consisted of an
equally spaced one-dimensional, 4 condition screen with 1 M
Hepes pH 7.5 and 1.15 M Ammonium Sulfate in the precipitate
solution held constant, and Jeffamine M-600 concentration
ranging from 11–14% v/v. The crystallization yields were
computed from the hits from 5 to 16 replicates trials for each
monoolein phase. While the RC preparation was capable of
producing crystals in solution with crystallization reagents
optimized for such growth [25], RC crystals did not form in the
absence of any monoolein lipid (not shown) using the precipitation
reagents employed.
4 X-ray diffraction of RC crystals
RC crystals were harvested directly from the wells and flash-
cooled in liquid Nitrogen without further cryoprotection. RC
crystals were subjected to maximum 30 second X-ray radiation
using an in house Rigaku FR-E+ Superbright X-ray generator,
Varimax HF optics, and a Rigaku Saturn 944+ detector. The
highest resolution X-ray diffraction spots were assigned manually
and were used to identify the resolution limit for each RC crystal
tested.
5 Incorporation experiments using the sandwich format
A portion of LCP was prepared as described [10] by mixing
monoolein with 44% (v/v) water at 16uC, yielding a transparent,
non-birefringent and highly viscous material. 400 nl of LCP were
dispensed into the center of a Laminex sandwich plate (Molecular
Dimensions, Suffolk, UK). Around the LCP slug 2.5 ml of RC
solution at 20 mg/ml were pipetted. A glass cover slip was
attached to seal the well and to establish contact of the protein
solution with the LCP. The well thickness was 100 micrometer.
The setup was placed under a Leica MZ12.5 microscope equipped
with an SPOT Insight 2MP Mosaic camera (Diagnostic
Instruments, Inc., Sterling Heights, MI) and illuminated with a
Volpi NCL 150 light source operated on the level 3 low setting.
After fixing exposure parameters and white balance, images were
recorded starting approx.K minute after assembly and then every
5 minutes for a total of ,16 hours. Images were analyzed using
ImageJ [35]. The scale in the images was approximated by using
the diameter of the well (5 mm) as a reference length. RC
concentrations were approximated by saturation levels that were
computed by employing RGB values and the ImageJ function
‘‘Save XY coordinates’’ using the formula saturation = (max-
min)/max?100. For each region of the image analyzed, a 144-pixel
area was selected and the corresponding saturation values were
averaged.
Supporting Information
Figure S1 Materials properties (transparency, birefrin-
gency, and viscosity) of the monoolein-based lipid
solutions employed in RC crystallization experiments
using the PLI approach. Different lipid phases were created in
syringe barrels by mixing solid monoolein with water. Water
content labels (w/v fractions) are used to align images and
tabulated data. A: Images of transilluminated syringe barrels with
clear and/or turbid materials. B: Images of syringe barrels
sandwiched between two crossed, linear polarizers (note that the
Membrane Protein Incorporation into Lipidic Phases
PLoS ONE | www.plosone.org 7 August 2011 | Volume 6 | Issue 8 | e24488

background between the barrels is black, indicating complete light
extinction). Lower Panel: Tabulated transparency ‘scores’ (N =
no, not transparent; Y = yes, transparent), birefringence ‘scores’
(N = no, not birefringent; Y = yes, birefringent; S = some
birefringence), and relative viscosity results. All lipid materials
utilized display properties that conform to materials used in
previous studies [ref] of monoolein phase behavior at room
temperature.
(TIF)
Figure S2 Representative X-ray diffraction results of
RC crystals grown by the PLI method. Shown are
screenshots with X-ray diffraction images representing initial
monoolein hydrations of 5% and 50%, depicting the best (A, B)
and worst (B, C) diffraction. In order to show low and high
resolution diffraction spots the diffraction images are shown in
pairs A, B and C, D, each with low and high contrast setting,
respectively. X-ray diffraction limits are listed in Fig. 3. Diffraction
images were acquired with a CCD area detector (Saturn 944+)
using a rotating copper anode X-ray source (Rigaku FR-E+).
Rotation range was 0.5 deg, exposure time 60 sec.
(TIF)
Acknowledgments
We thank C. Kors at ANL for assistance in the preparation of
crystallization-quality RC samples.
Author Contributions
Conceived and designed the experiments: EW DD PN. Performed the
experiments: EW DD JC. Analyzed the data: EW DD JC PN. Contributed
reagents/materials/analysis tools: EW DD. Wrote the paper: DD PN PDL
EW. Supplied RC materials: PDL.
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