Review

Structure and Functional

Characterization of Membrane
Integral Proteins in the Lipid Cubic
Phase

Dianfan Li 1 and Martin Caffrey 2

1 - CAS Center for Excellence in Molecular Cell Science, National Center for Protein Science Shanghai, Shanghai Institute of
Biochemistry and Cell Biology, Chinese Academy of Sciences, 333 Haike Road, Shanghai 201210, China
2 - Membrane Structural and Functional Biology Group, School of Medicine and School of Biochemistry and Immunology, Trinity
College Dublin, Dublin D02 R590, Ireland

Correspondence to Martin Caffrey and Dianfan Li: Dianfan Li is to be contacted at: Fax: þ86 21 2077 8212. Martin
Caffrey is to be contacted at: Fax: þ353 1 896 4253. dianfan.li@sibcb.ac.cn, martin.caffrey@tcd.ie.
https://doi.org/10.1016/j.jmb.2020.02.024
Edited by Filippo Mancia

Abstract
The lipid cubic phase (LCP) has been used extensively as a medium for crystallizing membrane proteins. It
is an attractive environment in which to perform such studies because it incorporates a lipid bilayer. It is
therefore considered a useful and a faithful biomembrane mimetic. Here, we bring together evidence that
supports this view. Biophysical characterizations are described demonstrating that the cubic phase is a
porous medium into and out of which water-soluble molecules can diffuse for binding to and reaction with
reconstituted proteins. The proteins themselves are shown to be functionally reconstituted into and to have
full mobility in the bilayered membrane, a prerequisite for LCP crystallogenesis. Spectroscopic methods
have been used to characterize the conformation and disposition of proteins in the mesophase. Procedures
for performing activity assays on enzymes directly in the cubic phase have been reported. Specific
examples described here include a kinase and two transferases, where quantitative kinetics and
mechanism-defining measurements were performed directly or via a coupled assay system. Finally, ligand-
binding assays are described, where binding to proteins in the mesophase membrane was monitored
directly by eye and indirectly by fluorescence quenching, enabling binding constant determinations for
targets with affinity values in the micromolar and nanomolar range. These results make a convincing case
that the lipid bilayer of the cubic mesophase is an excellent membrane mimetic and a suitable medium in
which to perform not only crystallogenesis but also biochemical and biophysical characterizations of
membrane proteins.
© 2020 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction and Background
The lipid cubic phase (LCP) has been in use now
for over two decades [1,2] as a medium in which to
crystallize integral membrane proteins. At this stage,
the LCP method is proven and robust with more than
700 records in the Protein Data Bank (PDB) to its
credit. Many of these represent high profile struc-
tures such as those of the G proteinecoupled
receptor (GPCR)-G protein complex [3], which
figured in the Nobel Prize in Chemistry in 2012 [4].
Other notables include the rhodopsin-arrestin com-
plex structure [5e7] and that of channelrhodopsin [8]
at the heart of the recent optogenetics revolution [9].
0022-2836/© 2020 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).
The method works with proteins covering the full
range of activities from enzymes, to transporters,
channels and receptors all the way to structural
proteins and complexes [2]. The focus of this study is
primarily on membrane enzymes. Of the PDB
records currently attributable to the in meso method,
94 refer to enzymes (Fig. 1). These range from
enzymes involved in lipid metabolism [10e19] and
post-translational modification [20e23] to oxidases
[24,25] and photosynthetic reaction centers [26,27].
The LCP is a liquid crystalline material. At its
simplest, it consists of lipid and water. The lipid most
commonly used for in meso crystallization trials is
the monoacylglycerol (MAG), monoolein (referred to
Journal of Molecular Biology (2020) 432, 5104e5123
Characterization of Membrane Proteins in the Lipid Cubic Phase 5105

Fig. 1. Membrane protein structures solved using LCP crystals. Numbers refer to counts of PDB records for the
indicated classes. The class “enzymes” includes lipid-metabolizing enzymes (46), post-translational modifying enzymes
(11), heme A synthases (2), nicotinamide nucleotide transhydrogenases (2), ferric ion reductases (2), respiratory oxidases
(15), photosynthetic reaction centers (15), and light-harvesting complexes (1). LCP ¼ lipid cubic phase; PDB ¼ Protein
Data Bank; GPCR ¼ G proteinecoupled receptor.

in the N.T MAG notation as 9.9 MAG 1) [28,29].
However, other shorter chain MAGs, such as 7.7
MAG, have been used effectively for growing
crystals [30], particularly of membrane protein
complexes [3,24]. The cubic phase has approxi-
mately equal parts of lipid and water. The lipid
adopts a familiar form, that of a lipid bilayer that is
hydrated on both sides. The packing density of the
mesophase is extraordinarily high with a surface
area of ~700 m 2/g [31]. This is achieved as a result
of the bilayer and the bathing aqueous channels on
either of its sides being continuous, folded, and
highly curved (Fig. 2). In consequence, the LCP is
referred to as being a bicontinuous or tricontinuous
mesophase. In terms of topology, the mid-plane of
the membrane takes the form of an (infinite) periodic
minimal surface [32,33].
1
MAGs are described in shorthand using the N.T notation, wherein N and
T refer, respectively, to the number of carbon atoms in the fatty acyl chain
on either side of the cis-olefin bond. Thus, 9.7 MAG corresponds to
monopalmitolein wherein the fatty acid, palmitoleic acid, is
(N þ T ¼ 9 þ 7 ¼ ) 16 carbon atoms long with a cis-double bond
between carbon atom numbers 9 and 10.
The principal lipid component of the mesophase is
referred to as the host lipid [2,11,34e36] to
distinguish it from additive lipids [11,37,38], usually
doped into the host lipid to give the mesophase
desirable physical, chemical, and, by extension,
functional properties. Cholesterol is an example of
an additive lipid. It is used extensively for in meso
crystallogenesis of GPCRs [2,39].
The cubic phase is just one mesophase that is
formed by MAG/water systems. Others include the
inverted hexagonal (HII) and the lamellar liquid
crystalline (La) phases as well as liquid and crystal-
line phases that form in a manner that is, at least,
temperature and composition dependent [28,29]. In
the simplest case, wherein the mesophase is made
of lipid and water, composition refers to their relative
amounts in the system. Phase behavior is conve-
niently and concisely conveyed in the form of a
temperature-composition phase diagram (Fig. 3)
that shows how different phases are stabilized at
different temperatures and compositions. In the case
of monoolein, the cubic phase forms at 20  C when 3
units (volume or weight) 2 of monoolein are
2
The mass density of monoolein is close to 1 at about 0.94 g/mL.
5106 Characterization of Membrane Proteins in the Lipid Cubic Phase
Fig. 2. A schematized view of the bicontinuous lipid
cubic mesophase. It consists of a highly curved, contin-
uous lipid bilayer, both sides of which are water coated. The
two water channels (blue and red) interpenetrate but never
contact one another because they are separated by a lipid
bilayer. A membrane protein reconstituted in the bilayer is
colored orange. Lipid components of the bilayer are shown
at the bottom left. The dimension of a unit cell within the
cubic phase (space group Im3m) is indicated in the top left.
Adapted from the study by Caffrey [45].
combined with 2 units of water. Note that along the
20  C isotherm, different mesophases, and indeed
the lamellar crystalline phase, form on reducing
water content. In the presence of excess water, a
two-phase system (cubic phase in equilibrium with
excess water or aqueous solution) emerges at ~40%
(w/w) water, reflecting the maximum water-carrying
capacity of the LCP.
To use the cubic phase for crystallization, the
membrane protein of interest must first be recon-
stituted into the lipid bilayer of the mesophase
[40,41]. This happens spontaneously as the pure
lipid and the protein solution are mixed to homo-
geneity forming the mesophase. The protein solution
is typically prepared using detergents. Fortunately,
many detergents are compatible with cubic phase
formation, provided they are not present at too high a
concentration [42,43]. Homogenization is achieved
in the space of a minute or two, typically at 20  C,
using a coupled syringe mixing device consisting of
two microsyringes connected by a coupler of narrow
bore [40,44]. Conditions are usually chosen so that
the pure cubic phase (by pure cubic phase, it means
the cubic phase in the absence of excess aqueous
solution or any other phase; see Fig. 3) is formed.
Evidence that this has been achieved includes the
production of an optically clear material that is
characteristically viscous, is nonbirefringent, and
has a distinct small-angle X-ray diffraction pattern
[28,41,45].
An assumption regularly made, and with some
reliability, is that the protein itself, and indeed the
other components of the protein solution, does not
alter significantly the mesophase in comparison with
using pure water. This is true in most circumstances.
However, in certain situations, a phase change does
occur. This has been documented for a range of
detergents when present at too high a concentration
[42,43,46]. We have also reported a temperature-
dependent transition to the HII phase with gramicidin
D, a pentadecapeptide proton channel, when used
at extremely high concentrations [47].
The goal of the in meso method is to grow crystals
of the target membrane protein for use in structure
determination. Crystallogenesis is proposed to come
about as a result of a local transition from the cubic to
the lamellar phase induced by equilibrating the so-
called precipitant solutions 3 with the protein-laden
mesophase [48,49]. In the process, the protein
preferentially partitions into the lamellar phase,
wherein it concentrates giving rise to a nucleus that
develops into a macroscopic crystal. The cubic
phase is tethered to the crystal by way of a lamellar
portal that serves as a conduit for proteins to migrate
from the bulk cubic phase. The cubic phase thus acts
as a reservoir to feed protein molecules to the face of
the growing crystal (Fig. 4). Elements of this
hypothesis for in meso nucleation and crystal growth
have been tested and are supported by experimental
observations [48,49]. The model is used to trouble-
shoot issues with and for rational approaches to
crystallization [3,5,24].
A feature often cited as an advantage of the in
meso method is that the bilayer of the LCP into which
the membrane protein is reconstituted as a prelude
to crystallogenesis is a good and a more natural
membrane mimetic, than a detergent micelle, as
used in more traditional crystallization methods. But,
is it? The LCP is composed of a lipid not commonly
found in membranes (a MAG, with or without additive
lipid); it is multiply branched, highly curved, and
densely packed [31]. To evaluate its usefulness as a
membrane mimetic and to establish (i) that the
protein survives the homogenization process, (ii)
that it is properly reconstituted, and (iii) that it is
structurally sound, mobile, and functional before
entering crystallization trials, it has been necessary
to investigate the protein's health (as in functional
3
A precipitant solution is used to trigger nucleation and subsequent growth
of crystals of macromolecules. Typical ingredients can include buffers
with different pH values and buffer identities and concentrations, salts,
polymers such as polyethylene glycol, and assorted small molecules
such as 2-methyl-2,4-pentanediol and glycerol.
Characterization of Membrane Proteins in the Lipid Cubic Phase 5107

Fig. 3. Miscibility of monoolein-water in temperature and composition space. Phase identification was made in the
cooling and heating directions from 20  C [28,29]. Phases are represented in cartoon form with water colored blue. At
temperatures lower than ~17  C, the liquid crystalline phases are metastable. HII ¼ inverted hexagonal phase; La ¼
lamellar liquid crystalline phase; Lc ¼ lamellar crystal phase; FI ¼ fluid isotropic phase; Cubic 1 ¼ cubic phase of space
group Pn3m; Cubic 2 ¼ cubic phase of space group Ia3d; Aq. ¼ aqueous solution. Adapted from the study by Caffrey and
Cherezov [41].

and conformational state and mobility) or otherwise
in situ. This is not necessarily easy to do because the
LCP is neither a liquid nor a solid. Rather, it is a
viscous and sticky liquid crystal with a texture
likened to that of thick toothpaste. In consequence,
it can be a challenge to handle. However, over the
years, tools, techniques, and materials
[2,40,41,44,51e62] have been developed such
that, with a little practice, the LCP can be manipu-
lated with relative ease. Fortunately, the mesophase
is porous with internal aqueous channels that are
continuous with the bathing solution in which it
resides. As a result, solutes can exchange by
diffusing back and forth between the aqueous
channels of the mesophase and the bathing solution
[51,63e65]. Because the cubic phase is bicontin-
uous, both sides of the membrane can be accessed
at once. Although this is an advantage in certain
applications, it is a disadvantage in others, in which
directionality is integral to functional characterization
as applies to transporters and channels. Fortunately,
the pure cubic phase is transparent, which means it
can be used for optical measurements including
spectroscopy. Another advantage of the LCP is that
it generally remains intact as a liquid crystalline bolus
in equilibrium with any excess aqueous solution
[28,46]. In other words, the mesophase does not
break up or go into solution, provided the concentra-
tion of the lipid in the system is sufficiently high. Such
a behavior relates to and is dictated by the under-
lying phase science of this liquid crystalline material,
as described in Fig. 3.
Before describing enzyme and other functional
assays that have been performed in the LCP, it is
important to show that (i) the mesophase itself is
porous to whatever water-soluble materials with
which we wish to perform the downstream assays,
(ii) the protein is reconstituted into the lipid bilayer of
the mesophase, (iii) the protein is free to move in that
membrane, and (iv) it has the characteristics of a
properly folded protein in situ in the mesophase. This
series of preliminary characterizations is described
first.
Biophysical Characterization of
Membrane Proteins in the LCP
Mobility in the aqueous channels of the meso-
phase
In the cubic phase before crystallogenesis, mem-
brane proteins reside distributed, presumably
5108
Fig. 4. Events proposed to take place during the
crystallization of membrane proteins in the lipid cubic
phase [49]. The process involves an initial reconstitution
into the bicontinuous bilayer of the mesophase (bottom left
quadrant). The added precipitant causes phase separa-
tion, in which proteins diffuse from the cubic phase by way
of a lamellar portal (upper left quadrant) to the advancing
face of the crystal (upper right quadrant). Cocrystallization
of the protein with lipid (cholesterol) is indicated. Dimen-
sions of the lipid (yellow oval with tail), detergent (pink oval
with tail), membrane or additive lipid (purple), protein (b2-
adrenergic receptor, blue; PDB code 2RH1) [50], and
bilayer and aqueous channels (dark blue) are drawn to
scale. The membrane is ~40-Å thick. Reproduced from the
study by Cherezov and Caffrey [48]. PDB ¼ Protein Data
Bank.
uniformly, throughout the full extent of the meso-
phase lipid bilayer [49]. That the protein, thus
reconstituted, is active can be tracked by monitoring
the consumption of substrate, production of product,
or by the binding or release of ligand. If these
assorted substrates, products, or ligands are water
soluble, their disappearance from or appearance in
the solution bathing the protein-laden mesophase
can be conveniently quantified, particularly when
they have strong ultraviolet (UV) and/or visible
absorbance or fluorescence. However, for the
assay to work, the mesophase must be truly porous
and with the aqueous channels continuous with the
bathing solution. In such a case, the spectroscopic
signature of the ligand can be tracked by monitoring
the signal in the bathing solution. This can be done
conveniently with a 96-well plate reader, as illu-
strated in Fig. 5. In this test case, the cubic phase,
prepared with an adenosine diphosphate (ADP)e
containing buffer [51], was placed in a 96-well plate
and was covered with an ADP-free buffer. ADP
release from the mesophase was tracked by
monitoring the increase in absorbance at 260 nm
of the bathing solution. ADP flooded out of the
mesophase, with the bulk of the nucleotide released
Characterization of Membrane Proteins in the Lipid Cubic Phase
in 10 min (Fig. 5b). This expected behavior shows
that transport as needed to assay a kinase or an
ATPase enzyme reconstituted in the cubic phase
should be possible. Furthermore, it shows that the
cubic phase is a porous medium.
Reconstitution
The assumption on which the in meso crystal-
lization method is based is that the integral
membrane protein target starts out reconstituted
into the bilayer of the mesophase on homogenizing
the protein solution with the host lipid. We have
demonstrated this to be the case with several test
proteins by means of fluorescence quenching
[47,51,53,54]. To this end, tryptophan fluorescence
was tracked as monoolein was replaced with
bromo-MAG, a quenching lipid. Bromo-MAG is a
MAG with bromine on carbons 9 and 10 of its acyl
chain. Monoolein and bromo-MAG are entirely
miscible and form the cubic phase over all
compositions as confirmed by small-angle X-ray
diffraction [54]. The quenching curve (Fig. 6) was
generated with diacylglycerol kinase [51], DgkA, a
homotrimer with 5 tryptophans in each monomer.
Quenching is as was observed with other mem-
brane proteins known to form crystals by the in
meso method [47,53,54]. This is convincing evi-
dence for reconstitution into the membrane of the
cubic phase, as expected.
Mobility in the membrane
Given the bicontinuous nature of the LCP, it is
expected that a reconstituted membrane protein
should be free to move in the membrane. This, of
course, is a requirement for crystallogenesis,
wherein the protein must migrate from the bulk
mesophase to a site of nucleation that evolves into a
macroscopic crystal. A protein that, for whatever
reason, is not mobile in the mesophase will not
crystallize.
Movement in the mesophase can be visualized
and tracked in different ways. In one simple
demonstration experiment, a bolus of mesophase
was prepared that had been doped with the lipophilic
dye, Sudan Red [66]. The dye resides solely in the
lipid bilayer of the mesophase, and the bolus is bright
red. Placing this next to a bolus of the mesophase
without any dye and that is colorless gives rise to
diffusion of the dye from one bolus to the other
(Fig. 7a). Tracking the profile of the dye along the
length of the sample as a function of time can be
used to estimate a diffusion coefficient for Sudan
Red in the membrane of the mesophase. This is a
very visual illustration of the fact that small, apolar
molecules can diffuse in the LCP. It forms the basis
for creating complexes of membrane proteins
Characterization of Membrane Proteins in the Lipid Cubic Phase 5109

Fig. 5. Characterizing the porous, sponge-like nature of the lipid cubic phase. (a) Cartoon representation of the
mesophase in the well of a microplate reader releasing water-soluble ADP into a bathing aqueous solution. The sticky,
viscous nature of the cubic phase is a distinct advantage in such studies. It remains adhering to the wall of the well and out
of the light path, wherein A260 measurements are performed throughout the release study. (b) Release of ADP from the
cubic phase tracked by following the change in time of A260 of the bathing solution [51]. The sample was prepared by
mixing monoolein with a solution of ADP. A volume of 0.005 mL of the mesophase was placed in a deep-well plate and
overlain with 0.2 mL of buffer. A260 of the bathing solution was tracked at 30  C by shaking in a microplate reader. The inset
shows the same measurement carried out in the absence of shaking. Solid, dashed, and dotted lines indicate technical
triplicates. Clearly shaking is important and is routine in all such studies. Panel b is reproduced from the study by Li and
Caffrey [51]. ADP ¼ adenosine diphosphate.

Fig. 6. Intrinsic fluorescence quenching of DgkA is consistent with reconstitution in the lipid cubic mesophase.
(a) Quenching of the enzyme's tryptophan fluorescence was brought about by replacing monoolein with the quenching
lipid, bromo-MAG. Fluorescence (Fc) was scaled to the nonquenched fluorescence (Fo) value recorded in the absence of
bromo-MAG [51]. (b) A model of DgkA (PDB ID 3ze5) [10] in the membrane with tryptophans identified by sticks with yellow
carbon atoms and sequence position. Quenching to the extent of 80% in neat bromo-MAG is consistent with the model.
Panel a is reproduced from the study by Li and Caffrey [51]. MAG ¼ monoacylglycerol; PDB ¼ Protein Data Bank.

reconstituted in the mesophase with apolar ligands
for structure determination work.
This same idea has been extended to testing the
mobility of light-harvesting complex II (LHII), a
multisubunit and pigment-bearing membrane pro-
tein [63]. Evidence for mobility is clearly shown in
the changing concentration profile along the length
of a mesophase bolus, as illustrated in Fig. 7b.
Similar observations have been made with the light-
driven proton pump, bacteriorhodopsin. This inten-
sely purple-colored protein can be “seen” by eye to
move in crystallization wells, in which a clear zone
surrounds purple-colored crystals, which is evi-
dence of depletion and migration of protein from
5110 Characterization of Membrane Proteins in the Lipid Cubic Phase

Fig. 7. Mobility in the membrane of the cubic mesophase. (a) Evidence for diffusion in the bilayer of the cubic phase
and in support of its fusogenic properties. Two mesophase boluses were brought into contact (blue arrow) in the barrel of a
microsyringe. One was doped with Sudan Red, a lipophilic dye (left of the arrow). The other (right of the arrow) had no dye.
The numbered panels show progress in the diffusion of the dye from the donor bolus (left) to the acceptor bolus (right) after
contact at elapsed times of (i) 0, (ii) 0.1, (iii) 0.2, (iv) 1.1, and (v) 5.2 days [66]. (b) Quantifying the migration of Sudan Red (i)
and LHII (ii) from a donor to an acceptor mesophase. The donor was either Sudan Red or the LHII-laden mesophase. The
acceptor was the mesophase without any additive. Quantitation along the length of the sample (x/L) was carried out
spectrophometrically and is reported in normalized units of concentration (Cx/L/Cx/L-0) [63]. (c) FRAP profiles for DgkA, a
small protein known to undergo in meso crystallization (solid line), and for a large integral membrane ion channel (dashed/
dotted lines) currently in crystallization trials. Measurements were performed using a fluorescence microscope in
mesophases composed of monoolein alone (solid and dashed lines) and with monoolein doped with 10 mol% cholesterol
(dotted line). The red and green arrows indicate when the photobleaching light was turned on and off, respectively.
Because the ion channel is as mobile in the pure monoolein mesophase as DgkA, mobility is unlikely to limit growing
crystals of this target protein by the in meso method. (d) Crystals of bacteriorhodopsin growing in 7.11 MAG. Regions of the
mesophase surrounding the crystals are less highly colored, consistent with protein diffusing from the mesophase for
incorporation into the crystal [77]. Reproduced from the study by Caffrey [66] (a), Clogston [63] (b), and Misquitta [77] (d).
LHII ¼ light-harvesting complex II; FRAP ¼ fluorescence recovery after photobleaching; MAG ¼ monoacylglycerol.

the bulk mesophase to the face of the growing

crystal (Fig. 7d) [66].
Nuclear magnetic resonance has been used to
monitor diffusion in the cubic phase [67,68]. While
more involved, the measurement can be carried out
relatively easily, and different types of diffusional
characteristics (rotation, translation) can be
investigated.
Diffusion in the membranes of whole cells and
tissues is now routinely performed by fluorescence
recovery after photobleaching (FRAP) [69e72]
(Fig. 7c). The process usually involves labeling the
protein with a fluorophore. This could be a small
molecule, such as fluorescein isothiocyanate [73] or
a fluorescent protein [74]. FRAP has also been used Fig. 8. MST of lysozyme in solution and in the lipid
to monitor protein mobility in the cubic mesophase cubic phase [79]. For the solution measurements (black
[75], with measurements of this type first reported in traces), the lysozyme concentration used was 20 mg/mL in
2001 [76]. We use a fluorescence microscope for Milli-Q water. In meso measurements (blue traces) were
low-throughput mobility screening by FRAP and only performed using the cubic phase prepared with monoolein
resort to it in cases where extensive crystallization at 40% (w/w) hydration using a lysozyme solution at a
trials have failed. It is important to note that the rate concentration of 50 mg/mL (the final lysozyme concentra-
of movement may not be overly important as far as tion in meso was 20 mg/mL). The temperature jump was
crystallogenesis is concerned, provided a good activated at time 0 s and was deactivated at time 31 s. Six
replicate samples were used for both sample types.
fraction of the protein in the bolus can and does Clearly, lysozyme undergoes positive thermophoresis in
diffuse. In fact, a slower moving protein may support bulk solution and within the aqueous channels of the cubic
more orderly nucleation and crystal growth, giving mesophase. Reproduced from the study by Wienken at al
rise to better quality crystals and structures. High- [78]. MST ¼ microscale thermophoresis.
Characterization of Membrane Proteins in the Lipid Cubic Phase 5111

throughput in meso FRAP screening instruments are
available commercially. However, these are extre-
mely expensive. Accordingly, we prefer to do
extensive crystallization screening first, which is
often possible and simpler to do because in meso
crystallization uses very little protein per trial. If we
fail to get crystal hits and are concerned the protein is
aggregated and/or immobile for whatever reason, a
quick FRAP measurement of the type described can
provide a definitive answer inexpensively and within
a few hours. Immobility would suggest that the
protein may need to be modified in some way,
possibly by mutational work, by choosing a different
ortholog, or by running a “buffer” optimization by
screening buffer type, concentration and pH, salt
type and concentration, detergent type and concen-
tration, and so on, to improve stability, homogeneity,
and solubility.
Microscale thermophoresis (MST) is a method
used to monitor ligand binding [78]. It is based on the
principle that macromolecules diffuse in response to
the imposition of a temperature gradient. The rate of
diffusion depends on the size, shape, and hydration
properties of the macromolecule that change on
ligand binding. Diffusion is monitored in either a
label-free mode using intrinsic tryptophan and/or
tyrosine fluorescence or using a fluorescently
labeled partner. We have shown that MST can be
performed with proteins in the cubic mesophase
(Fig. 8) [79]. In this case, the protein was the water-
soluble enzyme, lysozyme, which resides and
migrates (and crystallizes) [80] in the aqueous
channels of the mesophase. Clearly, the data show
that the protein diffuses in the mesophase in
response to a small jump in temperature. Because
MST instruments are relatively common, they
provide a very fast, simple, inexpensive, and
convenient way to gauge the mobility, and thus the
potential in meso crystallizability of any test mem-
brane protein, assuming it undergoes
thermophoresis.
Spectroscopic Measurements
When a protein contains pigment molecules that
play a role in function, the state of that protein in the
cubic mesophase can be assessed by means of
absorbance spectroscopy. The fact that the cubic
phase in pure form is optically clear makes such
spectroscopic measurements possible. The example
shown here is LHII [81], which plays a role in bacterial
photosynthesis. It has bacterial chlorophylls and
carotenoids as light-gathering pigments. The absor-
bance spectrum of the protein in detergent solution
and reconstituted into the bilayer of the cubic phase is
shown in Fig. 9a. They are remarkably similar to
features recognizable for both pigment types con-
sistent with a healthy protein. Similar measurements
have been performed with other pigment-containing
proteins such as bacteriorhodopsin [1], the photo-
synthetic reaction center [82], and visual rhodopsin
[83]. In the case of bacteriorhodopsin, the light cycle
has been investigated with crystals grown by the in
meso method [84e86]. With rhodopsin, photoactiva-
tion and coupling with transducin was demonstrated
directly in the cubic phase [83].
The UV absorbance and fluorescence character-
istics of tryptophan can be used to report on the
environment of that aromatic residue in a protein

Fig. 9. Absorption, fluorescence, and circular dichroism measurements on proteins in the cubic phase. (a)
Absorption spectrum of light-harvesting complex II in detergent solution (dashed line) and in the sponge phase (solid line
prepared with monoolein) [81]. (b) Spectrophotometry of OpcA in surfactant solution and in the cubic mesophase. Included
are the absorbance spectra of the protein in solution and in the cubic phase and fluorescence spectra of the protein in the
cubic phase with and without sialic acid. Excitation wavelengths of 280 nm and 305 nm were used [53]. (c) Circular
dichroism spectra of BtuB in solution and in the cubic mesophase. The spectrum below ~208 nm for the cubic phase is not
reliable because of strong background absorption. The data show that the secondary structure was not sensitive to
whether BtuB was in a detergent micelle or a bilayer environment [54]. Panel a is reproduced from the study by Cherezov
et al [81]. Panel b is reproduced from the study by Cherezov et al [53]. Panel c is reproduced from the study by Cherezov
et al [54]. CD ¼ circular dichroism.
5112 Characterization of Membrane Proteins in the Lipid Cubic Phase

[87e90]. Fortunately, the pure cubic phase is

optically clear, and the lipid used to make the
mesophase does not absorb strongly in the vicinity
of 280 nm and 340 nm, where tryptophan absorbs
and fluoresces maximally. Thus, it is possible to
record informative UV absorbance and fluorescence
spectra of membrane proteins reconstituted in the
cubic phase (Fig. 9b) [53]. These can be used to
report on the protein's disposition in the mesophase.
Most commonly, they serve to report on changes in
the protein in response to some perturbation, such
as contact, as described previously for quenching by
bromine in bromo-MAG, or the binding of a ligand, as
will be described belowin the following section.
Circular dichroism (CD) is used to report on the
secondary structure of a protein [91]. The useful
range in which CD measurements are performed
typically extends from 260 nm to 190 nm. Because
the lipid used to make the cubic phase has an
absorbance that is strong below about 215 nm [56],
useful CD measurements on proteins in the cubic
phase in this region are not possible. However, the
spectrum above 215 nm is measurable and can
Fig. 10. TLC analysis of the substrate for and
provide insight into the conformation of the protein, at product of DgkA acting on monoolein in the cubic
least in different states of dispersion, as shown for the phase [51]. Lane 1, monoolein. Lane 2, lysoPA. Lane 3,
vitamin B12ebinding protein, BtuB (Fig. 9c) [54]. The monoolein and lysoPA. Lane 4, reaction mix without DgkA.
minimum in the CD spectrum is characteristic of b- Lane 5, reaction mix plus DgkA. The reaction was run
sheet and is consistent with the known b-barrel overnight at 30  C. Lanes 4 and 5 show DgkA-dependent
structure of BtuB. These data support the view that conversion of the substrate to product (lane 5). The control
the protein retains a native conformation while (lane 4) illustrates that nonenzymatic phosphorylation of
reconstituted into the bilayer of the cubic mesophase. monoolein by ATP takes place at a low level. Reproduced
from the study by Li and Caffrey [51]. TLC ¼ thin layer
chromatography; lysoPA ¼ lysophosphatic acid; ATP ¼
adenosine triphosphate.
Functional CharacterizationdEnzyme
Activity
lysoPA, which is a potent surfactant. After prolonged
Kinase activitydDgkA activity where large quantities of monoolein are
converted to lysoPA, the mesophase eventually falls
DgkA is a kinase that functions as a homotrimer in apart as the surfactant dissolves the mesophase.
the bacterial cytoplasmic membrane [92]. It is However, in the early stages of the reaction, the
responsible for the adenosine triphosphate (ATP)e mesophase remains intact, and mechanistic enzy-
dependent phosphorylation of diacylglycerol forming mology studies can be performed with this sticky and
phosphatidic acid (PA) for use in phospholipid and viscous material.
membrane-derived oligosaccharide synthesis. The DgkA activity in the cubic phase was assayed in
enzyme is promiscuous in that it can use a number of two ways. In the first of these, the product of the
substrates other than diacylglycerols, including reaction, lysoPA, was monitored by thin layer
MAGs such as monoolein [51,93]. The structure of chromatography (TLC) [51]. The latter method
DgkA was solved using crystals grown by the in enables convenient separation of the substrate
meso method [10,94,95]. In preliminary work carried from the product on a TLC plate. Both can be
out while the crystal structure determination project visualized by wet ash staining. For the reaction to
was in train, DgkA was shown to be enzymatically take place, however, the ATP substrate must diffuse
active in the LCP [51]. Given that the enzyme is from the bulk bathing solution into and throughout
promiscuous and that it can use monoolein as a the enzyme-laden mesophase, wherein the enzyme
substrate, we chose to use monoolein both as the is surrounded (and saturated 4) by its lipid substrate,
host lipid creating the cubic mesophase membrane monoolein. That the mesophase is porous and that it
in which DgkA resides for crystallogenesis and as a
substrate with which to monitor its kinase activity. 4
The concentration of monoolein in the pure cubic mesophase is about
The product of the kinase reaction with monoolein is 2 M.
Characterization of Membrane Proteins in the Lipid Cubic Phase 5113

should support such water-soluble nucleotide mobi-
lity was convincingly demonstrated in the ADP
release study described earlier (Fig. 5). The data in
Fig. 10 show that indeed DgkA was active in the
mesophase, as evidenced by the formation of
lysoPA in a DgkA-dependent manner.
The second method used to monitor DgkA activity
in situ in the cubic mesophase was by way of a
coupled enzyme system [51,96]. This is a spectro-
photometric method that couples DgkA action with
sequential pyruvate kinase (PK) and lactate dehy-
drogenase (LDH) activity. Thus, for every mole of
ATP consumed and ADP produced by DgkA, one
mole of nicotinamide adenine dinucleotide hydrogen
(NADH) is consumed. The loss of NADH can be
conveniently monitored by tracking absorbance at
340 nm (A340) in a continuous manner using a
multiwell plate and a plate reader. For this purpose,
the DgkA-laden mesophase was included in the well
as an elongated thread-like bolus that, because the
mesophase is sticky, adheres naturally to the side-
wall of the well out of the way of the interrogating light
beam (Fig. 11). The reaction was initiated by adding
an aqueous reaction mix that included the coupling
enzymes, PK and LDH, ATP, and the relevant
substrates for the coupling enzymes for the reaction
to proceed. In control measurements performed with
DgkA in detergent solution and with dihexanoylgly-
cerol (DHG) as the lipid substrate and cardiolipin
(CL) as the activator, the reaction begins immedi-
ately without any lag (Fig. 11b). However, for DgkA
in the lipid mesophase, a well-defined lag in the
reaction progress curve is observed (Fig. 11c). This
is expected, given that time is needed for ATP to
diffuse into the mesophase for reaction and for the
product, ADP, to diffuse out for use by the coupling
enzymes in the bathing aqueous solution (Fig. 5).
The lag is followed by a linear progress curve
reflecting a steady state that, in turn, reflects the
catalytic turnover of the enzyme in the bilayer of the
mesophase (Fig. 11c). The linear region can be used
for initial rate calculations and for subsequent
kinetics profiling studies.
The specific activity (SA) of the kinase in detergent
solution with monoolein as the substrate was 27 U/
mg [51]. An operational SA of 17 U/mg was recorded
for DgkA reconstituted into the monoolein-based
mesophase under standard conditions (Fig. 11b).
Importantly, the two systems are distinctly different in
that one consists of a mixed micelle and the other
consists of a lipid bilayer. Thus, similar SAs are not
necessarily expected, given that the enzyme is more
than likely to function in an environment-specific
manner. Nonetheless, the fluorescence (Fig. 6) and
in meso activity data (Figs. 10 and 11c) demonstrate
that DgkA kinase is active while reconstituted into
the lipid mesophase.
Despite the complexity of the assay, it was
possible to characterize the kinetics of DgkA in the
bilayer of the mesophase. DgkA phosphorylates
diacylglycerol using Mg 2þ -ATP as the phosphate
donor. Monoolein also functions as the lipid sub-
strate. However, the concentration of monoolein in
the mesophase cannot be varied easily to determine
the corresponding Km and Vmax values. Presumably,
at 2 M lipid in the cubic phase, monoolein saturates
DgkA under conditions of assay. However, Mg 2þ-
ATP concentration can be varied, and how it affected

Fig. 11. Monitoring DgkA kinase activity in the cubic phase [51]. (a) The kinase activity was coupled to the oxidation
of NADH by the two enzymes, pyruvate kinase (PK) and lactate dehydrogenase (LDH), enabling convenient spectroscopic
monitoring of the assay at 340 nm. The cartoon shows how the assay is performed in a multiwell plate. (b and c) Reaction
progress in surfo and in meso at 30  C. In (b), the reaction was in surfo with the substrate, DHG, and cardiolipin, the
activator. In (c), the reaction was carried out with DgkA reconstituted in the mesophase formed by monoolein which
doubles as the substrate. The DgkA progress curve (solid line) is corrected (dashed line) for background changes
recorded without the enzyme (dotted line). Reproduced from the study by Li and Caffrey [51]. NADH ¼ nicotinamide
adenine dinucleotide hydrogen; DHG ¼ dihexanoylglycerol; ADP ¼ adenosine diphosphate; ATP ¼ adenosine
triphosphate; PA ¼ phosphatidic acid.
5114 Characterization of Membrane Proteins in the Lipid Cubic Phase

Fig. 12. Dependence of DgkA activity on the substrate, the activator, and protein concentration with the kinase
reconstituted in the cubic mesophase [51]. (a) Mg 2þ-ATP concentration dependence of DgkA kinase activity
(Lineweaver-Burk analysis for the determination of kinetic constants, Km and Vmax, is shown in the inset). (b) Mg 2þ
concentration dependence of kinase activity. (c) Enzyme concentration dependence of kinase activity recorded in a fixed
mesophase volume. The inset shows data points for the low enzyme loading region of the main plot. Reproduced from the
study by Li and Caffrey [51]. ATP ¼ adenosine triphosphate.

enzyme activity was quantified (Fig. 12a). Michaelis-
Menten kinetics was observed with Km and Vmax
values of 4 mM Mg 2þ-ATP and 14 U/mg recorded,
respectively [51]. Values of 1 mM Mg 2þ-ATP and 50
U/mg, respectively, were reported for DgkA in
detergent solution with the substrate DHG and the
activator CL [96]. In liposomes, the Km values range
from 0.2 to 1.8 mM [121]. Thus, reconstituted in the
cubic phase bilayer, DgkA functions as a well-
behaved enzyme.
DgkA in meso responded to Mg 2þ concentration
(Fig. 12b) as it did in surfo with diolein as the
substrate [97]. Accordingly, the standard in meso
assay mix included magnesium acetate at 55 mM.
It is possible that the lower Vmax value recorded in
the cubic phase can be accounted for by a fraction of
unfolded, dead enzyme. To address this, we showed
that a thermostabilized variant of DgkA, CLLD [98],
had the same relative activities in meso and in surfo
as the wild-type kinase [51]. Given that the CLLD

Fig. 13. Tracking PgsA converting CDP-DAG to PGP in the lipid cubic phase [51]. (a) Cartoon representation of the
reaction taking place in the porous mesophase. (b) Progress of the reaction monitored by following A272 due to release of
the CMP product from the mesophase into the bathing solution. The cubic phase was made by combining 0.08 mol %
(black) or 0.34 mol % CDP-DAG (red) in monoolein with the enzyme solution. The assay was performed at 37  C in an
assay mix containing 16 mM G3P (black, red). Control measurements carried out in the absence of PgsA (yellow), G3P
(green), or CDP-DAG (blue) were identical and show no activity. (c and d) Dependence of enzyme activity in the cubic
phase on G3P (c), and on the activator, MgCl2 (d), concentration. For G3P, 0 mM (black), 1 mM (red), and 16 mM (blue)
were tested. For MgCl2, 0 mM (black), 3 mM (red), and 100 mM (blue) were tested. Measurements were carried out with
0.17 mol% of CDP-DAG in monoolein in (c) and (d). Reproduced from the study by Li and Caffrey [51]. CDP-DAG ¼
cytidine diphosphate diacylglycerol; PGP ¼ phosphatidylglycerol phosphate; CMP ¼ cytidine monophosphate; G3P ¼
glycerol 3-phosphate.
Characterization of Membrane Proteins in the Lipid Cubic Phase
construct is highly stable [98], these results suggest
the reduced rates recorded in the cubic phase reflect
full enzyme activity and do not indicate denatured
DgkA. Consistent with this, we have obtained two
functionally relevant crystal forms of DgkA in meso.
The first form diffracted to 2.05 Å and showed
adenine-containing nucleotide-specific dissolution
on soaking [10], while the second form survived
the soaking and produced a structure of the complex
[94]. In addition, we have demonstrated in a later
study that DgkA can be refolded from acidic urea in a
detergent-depleted form to a catalytically functional
form in the cubic mesophase [99].
The DgkA activity rate was dependent on the
concentration of the enzyme in the cubic phase with
the rate increasing linearly initially, as expected
(Fig. 12c) [51]. This indicates that the kinase
behaves in meso as a classically functioning
enzyme. It is an important result because rates can
now be used under standard assay conditions to
reliably quantify active protein and substrate
concentrations.
Phosphatidyltransferase activitydPgsA
Phosphatidylglycerol phosphate (PGP) synthesiz-
ing enzyme, PgsA, plays a key role in bacterial
phospholipid synthesis [100]. This small membrane
enzyme converts cytidine diphosphate diacylgly-
cerol (CDP-DAG) and glycerol 3-phosphate (G3P)
to PGP and cytidine monophosphate (CMP). PGP is
subsequently converted to phosphatidylglycerol and
CL [101]. PgsA, has been assayed while reconsti-
tuted in the LCP (Fig. 13) [51]. The lipid substrate,
CDP-DAG, and product, PGP, remain in the bilayer
of the mesophase as their water-soluble counter-
parts, G3P and CMP, respectively, move in and out
of the enzyme-laden bolus (Fig. 13a). Because CMP
absorbs strongly at 272 nm, it is possible to
quantitatively track PgsA activity in the mesophase,
as was carried out for DgkA. In the case of PgsA,
however, the need for coupling reactions is not
necessary; the product CMP can be monitored
directly as it floods out of the mesophase into the
bathing aqueous solution for direct quantitation.
To monitor PgsA activity in meso, the enzyme was
reconstituted into the cubic phase as described
previously for DgkA, except that the hosting mono-
olein mesophase was predoped with the substrate
CDP-DAG (Fig. 13a). The concentration of the PgsA
solution used for mesophase preparation was
0.35 mg/mL. Ten microliters of the enzyme-laden
mesophase was dispensed in 1-mL boluses along
the sidewall of a well in a multiwell plate out of the
light path of the plate reader, as described previously
for DgkA. To begin the reaction, 0.2 mL of PgsA
assay mix (16 mM G3P, 0.2 mM TCEP, 0.1 mM
EDTA, 0.1 M MgCl2, 50 mM Tris/HCl) was added to
each well to cover the mesophase. The plate was
5115
placed in the microplate reader at 37  C, and A272
readings were recorded every 30 s for 100 min with
intermittent shaking. The data in Fig. 13bed
show that the kinetics of PgsA in the cubic
mesophase can be monitored quantitatively, directly
and continuously in medium-throughput fashion.
There is no need for labeling such as with radio-
isotopes or for cumbersome lipid extraction. The
assay method lends itself to library screening in
search of small molecules affecting PgsA activity
that might find application in design and discovery of
therapeutics.
Acyltransferase activitydPlsY
Phosphatidic acid is an essential intermediate in
phospholipid biosynthesis. It is formed by two
consecutive acylations of G3P. The first, catalyzed
by G3P acyltransferases, is known as the committed
step for phospholipid synthesis [100,101]. Two
distinct G3P acyltransferases, PlsB [102,103] and
PlsY [104], exist in bacteria. Unlike the conventional
type PlsB that uses thioesters (acyl-CoA or acyl-
carrier protein) as the acyl donor [105], PlsY uses an
unusual acyl donor, acyl phosphate (Acyl-P)
[104,106,107]. PlsY shares neither sequence nor
structure homology with other known acyltrans-
ferases, representing a unique class of enzyme.
PlsY exists ubiquitously in bacteria and is essential
for most gram-positive bacteria, many of which are
important human pathogens. Thus, PlsY is a
potential target for antibiotic development
[108,109], in support of which a high-resolution
structure was sought. The in meso method delivered
crystals [36] and a structure [13]. During the course
of the investigation, the enzyme was characterized
while reconstituted in the bilayer of the LCP [13] as a
prelude to crystallogenesis.
PlsY is relatively small (20.9 kDa) and is
extremely hydrophobic. Nearly 80% of its residues
are buried in the membrane. It transfers the acyl
moiety from Acyl-P to G3P, forming lysoPA and
inorganic phosphate (Pi) (Fig. 14). The Pi-releasing
activity of the enzyme can be monitored using a Pi
biosensor [110], the fluorescence of which
increases owing to conformational changes on Pi
binding. Initial attempts to assay PlsY in detergent
micelles failed because of the presence of the
contaminant Pi that was either carried over from the
chemical synthesis of Acyl-P or produced as a
result of Acyl-P degradation. Thus, Acyl-P was
“cleaned-up” before assay using the LCP itself as a
convenient medium for separating Pi from Acyl-P
based on their differential hydrophobicities/hydro-
philicities. To this end, PlsY and Acyl-P were
reconstituted into the LCP which was then applied
as adhering threads to the microplate wells as
carried out for DgkA and PgsA. Pi-free buffer was
added to soak and to rinse the LCP several times,
5116 Characterization of Membrane Proteins in the Lipid Cubic Phase

Fig. 14. Enzymatic assay of PlsY in the lipid cubic phase [13]. (a) Schematic representation of the PlsY assay
method. The assay works by detecting the product, inorganic phosphate (Pi), using a Pi sensor, a fluorescently labeled
phosphate-binding protein that shows enhanced fluorescence on ligand binding. LCP reconstituted with the enzyme PlsY
and its lipid substrate, Acyl-P, is dispensed on the sidewall of a microplate well. The LCP also contains trace amount of Pi
that need to be removed to avoid interference with the Pi biosensor. This is conveniently done by repeatedly soaking and
rinsing the sticky LCP with the Pi-free buffer. To initiate the reaction, the water-soluble substrate, G3P, was added to the
bathing solution. As demonstrated with ADP (Fig. 5), G3P diffuses into the water channels of the LCP, where it encounters
the enzyme. In consequence, Pi is produced and released from the LCP into the bathing solution, in which it binds the
sensor and causes an increase in fluorescence, which can be monitored continuously using a plate reader. (b) Progress
curves of the Pi biosensor assay. When G3P (magenta), 16:0-P (cyan), or enzyme (blue) was excluded, fluorescence
remained flat. When all the ingredients were included, fluorescence increased with time (black). An arrow indicates a 2-min
lag phase, reflecting the time required for the G3P to diffuse into the porous LCP from the bathing solution and for the Pi to
be released from the LCP. The slope of the short linear phase of the progress curve is used for activity calculation. The
leveling off in specific activity indicates product inhibition. (c) PlsY assay for lysoPA production using thin layer
chromatography. Lane 1, monoolein; lane 2, monoolein with lipid substrate, Acyl-P; lane 3, monoolein with the lipid
product, lysoPA; lane 4, monoolein with dodecyl maltoside; lane 5, reaction mix without the enzyme; lane 6, reaction mix
with the enzyme. (d and e) Michaelis-Menten and Lineweaver-Burk plot (insets) of the substrate concentration-activity
relationship. One unit (U) defines the enzymatic conversion of 1 mmol of substrates to products per minute. (f) Effect of
enzyme loading on the rate of activity. The inset shows data points for the low enzyme loading region of the main plot.
Results in (d)e(f) were from triplicate measurements. Reproduced from the study by Li et al [13]. Acyl-P ¼ acyl phosphate;
ADP ¼ adenosine diphosphate; DDM ¼ dodecyl maltoside; G3P ¼ glycerol 3-phosphate; LCP ¼ lipid cubic phase;
lysoPA ¼ lysophosphatic acid; MO ¼ monoolein.

during the course of which the contaminant Pi was
released and removed. By contrast, Acyl-P stayed
in the LCP because of the fatty acyl anchor. G3P,
together with the Pi sensor, was then added to the
bathing solution (Fig. 14a). The soluble substrate
diffuses into the LCP and triggers catalysis. The
product, Pi, is released from the LCP into the
bathing solution, increasing the fluorescence of the
biosensor. Fluorescence yield was monitored using
a plate reader at an excitation wavelength of
445 nm and emission wavelength of 500 nm.
Reaction progress curves show that the fluores-
cence of the Pi biosensor increased as a function of
time in a substrate and PlsY-dependent manner
(Fig. 14b). An initial lag phase reflects the time
required for the water-soluble substrate, G3P, to
diffuse into the porous mesophase and for the water-
soluble product, Pi, to diffuse out for detection. The
Characterization of Membrane Proteins in the Lipid Cubic Phase 5117

lag phase lasts about 2 min, which is less than that
observed with DgkA (compare Figs. 11c and 14b).
The disparity may derive from the size differential
and water solubility of G3P and Pi associated with
PlsY and ATP and ADP associated with DgkA. The
lag phase is followed by a short linear phase, which
was used to calculate initial rates. The in meso assay
was validated by TLC analysis. As shown in Fig. 14c,
lysoPA was detected in the mesophase loaded with
PlsY and Acyl-P after incubation with G3P. No lyso-
lipid product was observed in the absence of the
enzyme.
Using the coupled assay, the dependence on
substrate concentration of PlsY activity in the cubic
phase was investigated. PlsY displayed classic
Michaelis-Menten kinetics with a V m a x of
34.6 mmol min 1 mg 1 and apparent Km values of
0.1 mol% Acyl-P (relative to the host LCP lipid
monoolein) (Fig. 14d) and 1.4 mM G3P (Fig. 14e).
Furthermore, the rate scaled linearly with enzyme
concentration at low concentrations, consistent with
PlsY behaving as a catalyst (Fig. 14f). These results
indicate that PlsY is a well-behaved enzyme in the
LCP bilayer and that the LCP is a functionally
relevant membrane mimetic in which to perform
mechanistic enzymology studies as well as crystal-
lization, leading to structure determination.
Functional CharacterizationdLigand
Binding
Membrane proteins have myriad functions, and
not all are enzymes. When using the in meso method
to crystallize proteins that are not enzymes, it is
important to have available a means for evaluating
their functionality while reconstituted in the meso-
phase before crystallogenesis. The vitamin B12
(cyanocobalamin [CNCbl])etransporting protein,
BtuB, is one such protein. It is a b-barrel protein
that resides in the bacterial outer membrane
[111,112]. We used two approaches to gain insights
into its “state of health” in the cubic mesophase [54].
The first involved equilibrating a bolus of the BtuB-
laden mesophase with the buffer containing CNCbl.
Because CNCbl is pink, the bolus acquired a
distinctly pink color, whereas the reference meso-
phase with no BtuB remained colorless (Fig. 15a).
Furthermore, the loss of CNCbl from the bathing
solution to BtuB in the mesophase could be
monitored quantitatively by tracking its absorbance
at 361 nm, wherein CNCbl absorbs maximally.
Binding was also determined by measuring ligand-
induced quenching of intrinsic fluorescence in BtuB
[54]. To this end, the protein was reconstituted in the
cubic mesophase using a solution of apo-BtuB in
0.1% (w/v) lauryldimethylamine oxide. The BtuB-
laden cubic phase was combined with a solution
containing 0e10 mM CNCbl. Fluorescence was
recorded in the 320- to 360-nm region at an
excitation wavelength of 305 nm. Control measure-
ments were carried out with apo-BtuB in detergent
solution. Kd values were determined by Scatchard
analysis [113]. The extent of quenching with ligand
saturation was ~30% for protein in solution and in the
cubic mesophase. Binding was extremely tight with a
Kd of ~1 nM recorded for BtuB in solution and in the
mesophase (Fig. 15b). Similar Kd values have been
reported for the native membrane-bound and solu-
bilized forms of BtuB. It is apparent therefore that
BtuB reconstitutes into the bilayer of the cubic phase
in an active form before in meso crystallization.

Fig. 15. Ligand binding to membrane proteins in the lipid cubic phase [54]. (a) Binding to BtuB of CNCbl (pink
color). A bolus of cubic phase with (i and iii) and without (ii) reconstituted BtuB equilibrated at 20  C for 6 days with a
solution of 0.07 mM CNCbl. In (iii), the bathing solution was replaced with the ligand-free buffer just before the photograph
was taken, so the labeling of the bolus is more apparent. The bolus can be seen as an egg-shaped object at the bottom of
the cuvettes. (b) Scatchard analysis for BtuB/CNCbl binding in detergent solution (solid circles) and in the cubic
mesophase (open circles). The corresponding Kd values were 1.02 and 1.24 nM. (c) Scatchard analysis for OpcA/sialic
acid (SA) binding in solution and in the cubic mesophase; the Kd values were 0.6 and 0.4 mM, respectively. Reproduced
from the study by Cherezov et al [54] (a and b) and Cherezov et al [53] (c). CNCbl ¼ cyanocobalamin.
5118 Characterization of Membrane Proteins in the Lipid Cubic Phase
A similar analysis to that just described for BtuB
has been performed with the sialic acidebinding
protein, OpcA, an adhesin in the bacterial outer
membrane [53]. Sialic acid quenches intrinsic OpcA
fluorescence to the extent of 20% at saturation. This
degree of quenching was used to measure a Kd
value for OpcA in the cubic phase of ~1 mM sialic
acid, which is similar to the value obtained for the
protein in detergent micelles (Fig. 15c).
Perspectives
The results presented here support the view that
the continuous and curved bilayer of the cubic phase
is a reasonable biomembrane mimetic. In what
follows, we summarize some of the features of the
mesophase that distinguishes it from other mem-
brane mimetics such as detergent micelles, bicelles,
nanodiscs, and unilamellar vesicles.
The cubic phase is viscous and sticky. This
unique rheology sets it apart from other mimetics
which, under conditions of routine use, all form
liquid solutions or dispersions. As a result, they are
easily prepared and manipulated. But likewise, with
the right tools, the viscous cubic phase can be
readily handled. Thus, the coupled syringe mixer
enables mesophase preparation in a controlled
environment [44]. The loaded microsyringe facil-
itates accurate and precise dispensing of meso-
phase boluses down to subnanoliter volumes for
crystallization and functional screening [59,62,114]
and for the delivery of continuous “jets” for serial
crystallography at synchrotron [115e117] and free-
electron laser X-ray facilities [118]. The viscous and
sticky nature of the mesophase means also that
when it is placed on a solid surface, such as in the
base of a well for crystallization or the side of a well
for functional assay, the bolus stays put. For the
most part, it does not move or fragment even when
subjected to shaking or mixing as part of the assay
protocol.
The cubic phase spontaneously and macroscopi-
cally phase separates to coexist in equilibrium with
the bulk aqueous solution (Fig. 3). This property
reflects the fact that, as a liquid crystal, the cubic
phase follows Gibbs phase rule [119]. Accordingly,
when its water-carrying capacity is exceeded, the
added aqueous medium appears as a second
coexisting and physically separable phase. This
feature is the basis of the in meso crystallization
method, where a fully hydrated bolus of the protein-
laden mesophase equilibrates with a bathing pre-
cipitant solution. It is also exploited for membrane
protein renaturation [99] and in the cubicon method
for the stepwise ramping up of protein concentration
in the cubic phase using a protein solution that fails
to concentrate by other, more traditional methods
[46].
The cubic phase has been likened to a nanopor-
ous molecular sponge. The analogy to a domestic
sponge is a good one; it posits that the fabric of the
sponge corresponds to the continuous, multiply
branched lipid bilayer of the cubic phase while the
water-filled pores in the sponge are analogous to the
pair of interpenetrating but noncontacting aqueous
channels that enmesh one another and that perme-
ate the mesophase. Porosity is the key to all
applications of the cubic phase that includes its
functioning as a membrane mimetic. Thus, the
ingredients of a bathing solutiondwater, substrates,
ligands, salts, buffers, additives, and so ondcan all
diffuse into the interior of the bolus at rates that differ
depending on the activity (concentration) gradient,
aqueous solubility, partitioning into and on the lipid
bilayer, size, and shape. The diffusion rate also
depends on channel pore size and tortuosity and on
the composition of the lipid bilayer. It influences the
outcome of a crystallization trial and the evolving
local composition of the aqueous channels that, in
turn, affect the conformation and activity of recon-
stituted proteins.
In pure form, the cubic mesophase is optically
clear. This is generally the case for the other
mimetic systems with which the cubic phase is
being compared. Optical clarity is an invaluable
feature that facilitates the detection of the smallest
in meso crystals as initial hits, which when
observed turns a project from one concerned
with a level of blind screening into a well-focused
and directed optimization campaign. Optical clarity
also means that spectroscopic methods can be
brought to bear on proteins reconstituted in the
mesophase as a means for quantifying in situ
conformational state, mobility, stability, and func-
tional activity.
The cubic phase is optically isotropic. It is therefore
nonbirefringent. This is of use for detecting crystals
in and harvesting them from the cubic phase.
Crystals are often birefringent and thus appear as
bright, sometimes colored, objects on a dark (cubic
phase) background when viewed with under a
polarized light microscope. Isotropic also means
that the bilayer of the cubic phase lacks sidedness.
Thus, proteins reconstitute randomly orientated
across the membrane. Although this is not a problem
for crystallogenesis where polar and apolar crystals
can form regardless, it constitutes an obstacle for
investigating net vectoral transport in meso. Thus,
for example, an ion transported by protein molecule
A oriented in one direction across the membrane can
be transported by an adjacent or indeed a distant
protein molecule B oriented in the opposite direction
such that no net ion transport is detected. In this way,
Characterization of Membrane Proteins in the Lipid Cubic Phase
then, the cubic phase is analogous to micelles,
bicelles, and nanodiscs, which, in routine use, have
no defined sidedness. The membrane of a uni-
lamellar liposome, however, has sidedness that
makes bilayered vesicles indispensable for vectoral
transport studies.
In the cubic phase, both sides of the membrane
are accessible by water-soluble substances that
can enter and diffuse throughout its aqueous
channels. This property arises because the cubic
phase is bicontinuous. It has a single continuous
apolar compartment defined by the lipid bilayer
and a pair of continuous polar aqueous compart-
ments, one on either side of the bilayer. Therefore,
a water-soluble ligand, for example, can passage
into and throughout one of the channels to bind to
one of the extramembrane domains of a trans-
membrane receptor. The signal generated can be
relayed through the protein to the extramembrane
domain on the opposite side of the membrane. If a
downstream partner has been included in the
bathing solution and enters the channels, it can
now bind to the ligand-activated receptor. This is
analogous to what is possible with other “open”
mimetics such as micelles, bicelles, and nano-
discs. However, it is distinctly different from an
empty liposome, in which a protein reconstituted
with an outside-out orientation in the membrane
can only bind the ligand.
The density of membrane in the cubic phase is
extraordinarily high. By mass, and indeed by
volume, half of the cubic phase is composed of a
bilayered membrane. The other half is an aqueous
solution divided equally between the two sides of the
bilayer. The concentration of the lipid, monoolein, in
the pure cubic phase approaches 2 M. Therefore, a
protein reconstituted to 0.1 mol% with respect to the
lipid is already at a concentration of 2 mM in the
mesophase. Such a high local concentration can be
an advantage in that the signal from the protein, be
that in the form of ligand binding, enzyme turnover,
or a spectroscopic feature, is higher than that
available with routine preparations of other mem-
brane mimetics.
The aqueous channels in the cubic phase are
narrow and tortuous; they bend and twist (Fig. 2).
Thus, diffusion rates are less than those in bulk
solution or a dispersion, as would prevail with other
membrane mimetics. A consequence of a reduced
diffusion rate was the anticipated and observed lag
period in the progress of reactions catalyzed by
enzymes reconstituted in the cubic phase (Figs. 11,
13 and 14). Thus, there is an initial delay for water-
soluble substrates to diffuse throughout the bulk of
the mesophase and for products to diffuse back out
for detection. It is only after the initial lag that a
necessarily transient, steady-state condition is
5119
established that can be used to gauge the intrinsic
activity of the enzyme.
A particularly appealing feature of the cubic phase
exploited in some of the functional assays included
in this review is the fact that, with the right substrates
and products, it is not necessary to perform an
additional step to physically separate the two for
detection and quantitation. In the case of PgsA, its
lipid substrate, CDP-DAG, remained in the cubic
phase on the side of the well, whereas the water-
soluble product, CMP, diffused out for detection as
the water-soluble substrate, G3P, diffused into the
mesophase for reaction (Fig. 13). A similar reaction
type is carried out by PlsY, which likewise has water-
soluble and lipid substrates and products (Fig. 14).
With PlsY, interfering Pi carried into the mesophase
adventitiously was removed by a simple on-plate
soaking/washing procedure, exploiting the sticky,
viscous, phase-separated, and porous nature of the
cubic phase.
The cubic phase is an attractive medium in which
to crystallize membrane proteins because it incor-
porates a bilayered membrane. Accordingly, the
target protein enters the crystal lattice from an
environment analogous to the one it calls home in
the cell from which it came. This distinguishes the
cubic phase from detergent micelles that are
commonly used for membrane protein crystalliza-
tion. The sense therefore is that the structure of a
target protein obtained by the in meso method is
likely to more faithfully reflect its native state. As
noted, the cubic phase is close to 2 M in lipid
(monoolein) concentration. This is an extraordinarily
high lipid concentration. Indeed, in many structures
obtained with -grown crystals grown in meso,
structured monoolein decorates the surface of the
protein, often recapitulating a lipid bilayer as found in
a native membrane. Once again, this provides
reassurance that the structure observed is a
native-like one. An added bonus of the high lipid
concentration in the hosting mesophase is that lipid
molecules often show up bound to the protein at
sites that make perfect sense. By doing so, they help
identify natural lipid-binding sites in the case of lipid-
metabolizing and lipid-binding proteins. This was the
case with the three enzymes in the bacterial
lipoprotein post-translational modifying pathway
[20,21,120]. It was also observed with GPCRs,
wherein functional and stabilizing cholesterol is
present as part of cocomplex structures, obtained
using the mesophase doped with 10 wt% cholesterol
(www.gpcrdb.org). There is a possible downside,
however, in that attempts to obtain structures of
proteins bound to native lipids or to lipid-like or
hydrophobic ligands may have to compete with the
hosting lipid, which is present at a very high
concentration.
5120 Characterization of Membrane Proteins in the Lipid Cubic Phase
In sum, it is clear that the cubic mesophase is a
generally useful system to investigate the biochem-
ical and biophysical properties of membrane integral
proteins and that, despite its challenging rheology, it
is a versatile and a tractable nanoporous biomaterial.
The mesophase can be used in multiwell plates and
is suited to inexpensive, parallel, and medium-
throughput screening of the type that should facilitate
future crystallization, structure function, and drug
design and discovery studies.
Acknowledgments
The authors thank synchrotron facility scientists at
the Advanced Photon Source, Diamond Light
Source and the Swiss Light Source for assistance
and support, and past and present members of the
Membrane Structural and Functional Biology group
for assorted contributions to the study. This work
was supported in part by Science Foundation Ireland
award 16/IA/4435 (M.C.) and by the National Natural
Science Foundation of China award 31870726
(D.L.).
Conflict of interest
None.
Received 4 January 2020;
Received in revised form 14 February 2020;
Accepted 19 February 2020
Available online 27 February 2020
Keywords:
In meso method;
Kinetics;
Mechanism;
Membrane enzyme;
X-ray crystal structure
Abbreviations used:
Acyl-P, acyl phosphate; ADP, adenosine diphosphate;
ATP, adenosine triphosphate; CD, circular dichroism;
CDP-DAG, cytidine diphosphate diacylglycerol; CL, car-
diolipin; CMP, cytidine monophosphate; CNCbl, cyano-
cobalamin; DHG, dihexanoylglycerol; DM,
decylmaltoside; EDTA, ethylenediaminetetraacetic acid;
FRAP, fluorescence recovery after photobleaching; FITC,
fluorescein isothiocyanate; GPCR, G proteinecoupled
receptor; G3P, glycerol 3-phosphate; HEPES, (4-(2-
hydroxyethyl)-1-piperazineethanesulfonic acid; LDH, lac-
tate dehydrogenase; LHII, light-harvesting complex II;
LCP, lipid cubic phase; MAG, monoacylglycerol; MO,
monoolein; MST, microscale thermophoresis; NADH,
nicotinamide adenine dinucleotide hydrogen; PA, phos-
phatidic acid; PG, phosphatidylglycerol; PGP, phosphati-
dylglycerol phosphate; PK, pyruvate kinase; TCEP, tris(2-
carboxyethyl)phosphine; TLC, thin layer chromatography;
UV, ultraviolet.
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