Structure and Functional Characterization of Membrane Integral Proteins in the Lipid Cubic Phase

Journal Pre-proof Structure and Functional Characterization of Membrane Integral Proteins in the Lipid Cubic Phase Dianfan Li, Martin Caffrey PII:

S0022-2836(20)30201-1

DOI:

https://doi.org/10.1016/j.jmb.2020.02.024

Reference:

YJMBI 66463

To appear in:

Journal of Molecular Biology

Received Date: 4 January 2020 Revised Date:

14 February 2020

Accepted Date: 19 February 2020

Please cite this article as: D. Li, M. Caffrey, Structure and Functional Characterization of Membrane Integral Proteins in the Lipid Cubic Phase, Journal of Molecular Biology, https://doi.org/10.1016/ j.jmb.2020.02.024. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 The Author(s). Published by Elsevier Ltd.

Graphical abstract

Synopsis. A cartoon representation of a glycerolipid metabolizing enzyme, PgsA, reconstituted into the bilayer of a nanoporous membrane mimetic, the lipidic cubic phase. The enzyme is shown transferring part of a membrane-associated substrate, CDP-DAG, to a water-soluble substrate, G3P, creating the membrane-bound PGP and water-soluble CDP. PgsA activity can be monitored directly and continuously as the rate of CMP production using miniscule amounts of enzyme in high-throughput multi-well microplates.

Page 1 of 28 Structure and Functional Characterization of Membrane Integral Proteins in the Lipid Cubic Phase Dianfan Li,1 Martin Caffrey2 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.

Corresponding Authors: Dianfan Li. [email protected] (+86 21 2077 8212) Martin Caffrey. [email protected] (+353 1 896 4253)

Declarations of interest: none

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 pre-requisite 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 made directly or via a coupled assay system. Finally, ligand binding assays are described where binding to proteins in the mesophase membrane were 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.

Keywords In meso method; Kinetics; Mechanism; Membrane enzyme; X-ray crystal structure

Page 2 of 28 Abbreviations Acyl-P, acyl-phosphate; ADP, adenosine di-phosphate; ATP, adenosine tri-phosphate; CD, circular dichroism; CDP-DAG, cytidine diphosphate-diacylglycerol; CL, cardiolipin; CMP, cytidine monophosphate; CNCbl, cyanocobalamin; DHG, dihexanoylglycerol; DM, decylmaltoside; EDTA, ethylenediaminetetraacetic acid; FRAP, fluorescence recovery after photobleaching; FITC, fluorescein isothiocyanate; GFP, green fluorescent protein; GPCR, G-protein coupled receptor; G3P, glycerol 3-phosphate; HEPES, (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; LDAO, lauryldimethylamine oxide; LDH, lactate dehydrogenase; LHII, light-harvesting complex II; LCP, lipid cubic phase; MAG, monoacylglycerol; MO: monoolein; MST, microscale thermophoresis; NADH, nicotinamide adenine dinucleotide hydrogen; PA: phosphatidic acid; PG, phosphatidylglycerol; PGP, Phosphatidylglycerol phosphate; PK, pyruvate kinase; TCEP, tris(2-carboxyethyl)phosphine); TLC, thin layer chromatography; UV, ultraviolet.

Cover image – please consider Graphical Abstract

Page 3 of 28 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 over 700 records in the Protein Data Bank (PDB) to its credit. Many of these represent high profile structures such as that of the GPCR-G protein complex [3] which figured in the Nobel Prize in Chemistry in 2012 [4]. Other notables include the rhodopsin-arrestin complex structure [5-7] and that of channelrhodopsin [8] at the heart of the recent optogenetics revolution [9]. 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 paper is primarily on membrane enzymes. Of the PDB records currently attributable to the in meso method, 94 refer to enzymes (Figure 1). These range from enzymes involved in lipid metabolism [10-19] and posttranslational modification [20-23] to oxidases [24, 25] and photosynthetic reaction centers [2628].

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 metabolising enzymes (46), posttranslational 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).

Page 4 of 28 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 in the N.T MAG notation as 9.9 MAG1) [29, 30]. However, other shorter chain MAGs, such as 7.7 MAG, have been used effectively for growing crystals [31], particularly of membrane protein complexes [3, 24]. The cubic phase is approximately equal parts lipid and water. The lipid adopts a familiar form, that of a lipid bilayer which is hydrated on both sides. The packing density of the mesophase is extraordinarily high with a surface area of ~700 m2/g [32]. 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 (Figure 2). In consequence, the LCP is referred to as being a bi- or tri-continuous mesophase. In terms of topology, the mid-plane of the membrane takes the form of an (infinite) periodic minimal surface [33, 34].

Fig. 2. A schematized view of the bicontinuous lipid cubic mesophase. It consists of a highly curved, continuous 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 ref. 46. The principal lipid component of the mesophase is referred to as the host lipid [2, 11, 35-37] to distinguish it from additive lipids [11, 38, 39], 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, 38, 40]. 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 (Lα) phases as well as liquid and crystalline phases that form in a manner that is, at least, temperature and composition dependent [29, 30]. In the simplest case where the mesophase is made of lipid and water, composition refers to their relative amounts in the system. Phase behaviour is conveniently and concisely conveyed in the form of a temperature-composition phase diagram (Figure 3) which 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 combined with 2 units of water. 1

MAGs are described in shorthand using the N.T notation where 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 where the fatty acid, palmitoleic acid, is (N + T = 9 + 7 =) 16 carbon atoms long with a cis double bond between carbon atom numbers 9 and 10. 2 The mass density of monoolein is close to 1 at about 0.94 g/mL

Page 5 of 28 Note that along the 20 °C isotherm different mesophases, and indeed the lamellar crystalline (Lc) phase, form upon 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 lipid cubic phase.

Fig. 3. Miscibility of monoolein-water in temperature and composition space. Phase identification was made in the cooling and heating directions from 20 °C [29, 30]. Phases are represented in cartoon form with water colored blue. Below ∼17 °C the liquid crystalline phases are metastable. Abbreviations: HII, inverted hexagonal phase; Lα, lamellar liquid crystalline phase; Lc, lamellar crystal phase; FI, fluid isotropic phase; Cubic 1, cubic phase of space group Pn3m; Cubic 2, cubic phase of space group Ia3d; Aq., aqueous solution. Adapted from ref. 42. To use the cubic phase for crystallization, the membrane protein of interest must first be reconstituted into the lipid bilayer of the mesophase [41, 42]. This happens spontaneously as the pure lipid and the protein solution are mixed to homogeneity 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 [43, 44]. 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 [41, 45]. Conditions are usually chosen so that the pure cubic phase (by pure cubic phase is meant cubic phase in the absence of excess aqueous solution or any other phase; see Figure 3) is formed. Evidence that this has been achieved includes the production of an optically clear material that is characteristically viscous, is non-birefringent and has a distinct small-angle X-ray diffraction pattern [29, 42, 46].

Page 6 of 28 An assumption regularly made, and with some reliability, is that the protein itself, and indeed the other components of the protein solution, do not alter significantly the mesophase in comparison to 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 [43, 44, 47]. 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 [48]. 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 so-called precipitant solutions3 with the protein-laden mesophase [49, 50]. In the process, the protein preferentially partitions into the lamellar phase where 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 which 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 (Figure 4). Elements of this hypothesis for in meso nucleation and crystal growth have been tested and are supported by experimental observations [49, 50]. The model is used to trouble-shoot issues with and for rational approaches to crystallization [3, 5, 24].

Fig. 4. Events proposed to take place during the crystallization of membrane proteins in the lipid cubic phase [50]. The process involves an initial reconstitution into the bicontinuous bilayer of the mesophase (bottom left quadrant). Added precipitant causes phase separation where 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). Co-crystallization of the protein with lipid (cholesterol) is indicated. Dimensions of the lipid (yellow oval with tail), detergent (pink oval with tail), membrane or additive lipid (purple), protein (β2-adrenergic receptor, blue; PDB code 2RH1) [51], and bilayer and aqueous channels (dark blue) are drawn to scale. The membrane is ~40 Å thick. Reproduced from ref. 49. 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, compared to 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 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.

Page 7 of 28 or without additive lipid), it is multiply branched, highly curved and densely packed [32]. 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 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 oft likened to that of a thick toothpaste. In consequence, it can be a challenge to handle. However, over the years tools, techniques and materials [2, 41, 42, 45, 52-63] have been developed such that, with a little practice, the LCP can be manipulated 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 [52, 64-66]. Because the cubic phase is bicontinuous both sides of the membrane can be accessed at once. While this is an advantage in certain applications, it is a disadvantage in others where directionality is integral to functional characterization as applies with 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 [29, 47]. In other words, the mesophase does not break up or go into solution provided the concentration of lipid in the system is sufficiently high. Such behaviour relates to and is dictated by the underlying phase science of this liquid crystalline material, described in Figure 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 mesophase. In the cubic phase prior to crystallogenesis, membrane proteins reside distributed, presumably uniformly, throughout the full extent of mesophase lipid bilayer [50]. That the protein, so 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 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 illustrated in Figure 5. In this test case, the cubic phase, prepared with an ADP-containing buffer [52], was placed in a 96-well plate and was covered with 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 in 10 min (Figure 5b). This expected behaviour shows that transport as needed to assay a kinase or an ATPase enzyme reconstituted in the cubic phase should be possible. Further, it shows that the cubic phase is a porous medium.

Page 8 of 28 Reconstitution. The assumption upon which the in meso crystallization method is based is that the integral membrane protein target starts out reconstituted into the bilayer of the mesophase upon 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 [48, 52, 54, 55]. To this end, tryptophan fluorescence was tracked as monoolein was replaced by bromo-MAG, a quenching lipid. Bromo-MAG is a MAG with bromine on carbons 9 and 10 of its acyl chain. Monoolein and bromoMAG are entirely miscible and form the cubic phase over all compositions as confirmed by smallangle X-ray diffraction [55]. The quenching curve (Figure 6) was generated with diacylglycerol kinase [52], DgkA, a homo-trimer with 5 tryptophans in each monomer. Quenching is as was observed with other membrane proteins known to form crystals by the in meso method [48, 54, 55]. This is convincing evidence for reconstitution into the membrane of the cubic phase, as expected.

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 where A260 measurements are made 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 [52]. The sample was prepared by mixing monoolein with a solution of ADP. 0.005 mL of the mesophase were placed in a deep-well plate and overlain with 0.2 mL buffer. A260 of the bathing solution was tracked at 30 °C with 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 ref. 52. 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 where 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 [67]. The dye resides solely in the lipid bilayer of the mesophase and the bolus is bright red. Placing this next to a bolus of mesophase without any dye and that is colorless gives rise to diffusion of the dye from one bolus to the other (Figure 7a). Tracking the profile of dye along the length of the sample as a function of time can be used to estimate a diffusion coefficient

Page 9 of 28 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 reconstituted in the mesophase with apolar ligands for structure determination work.

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 non-quenched fluorescence (Fo) value recorded in the absence of bromo-MAG [52]. (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 ref. 52. This same idea has been extended to testing the mobility of the light harvesting complex (LHII), a multi-subunit and pigment bearing membrane protein [64]. Evidence for mobility is clearly shown in the changing concentration profile along the length of a mesophase bolus, as illustrated in Figure 7b. Similar observations have been made with the light driven proton pump, bacteriorhopsin. This intensely purple colored protein can be ‘seen’ by eye to move in crystallization wells where a clear zone surrounds purple colored crystals, evidence of depletion and migration of protein from the bulk mesophase to the face of the growing crystal (Figure 7d) [67]. Nuclear magnetic resonance has been used to monitor diffusion in the cubic phase [68, 69]. Whilst 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 using fluorescence recovery after photobleaching (FRAP) [70-73] (Figure 7c). The process usually involves labelling the protein with a fluorophore. This could be a small molecule, such as fluorescein isothiocyanate (FITC) [74] or a protein like Green Fluorescent Protein (GFP) [75]. FRAP has also been used to monitor protein mobility in the cubic mesophase [76] with measurements of this type first reported on in 2001 [77]. We use a fluorescence microscope for low throughput mobility screening by FRAP, and only resort to it in cases where extensive crystallization trials have failed. It is important to note that the rate of movement may not be overly important as far as crystallogenesis is concerned provided a good fraction of the protein in the bolus can and does diffuse. In fact, a slower moving protein may

Page 10 of 28 support more orderly nucleation and crystal growth giving rise to better quality crystals and structures. High throughput in meso FRAP screening instruments are available commercially. However, these are extremely 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, choosing a different orthologue or by running a ’buffer’ optimization by screening buffer type, concentration and pH, salt type and concentration, detergent type and concentration, etc., to improve stability, homogeneity and solubility.

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 arrow). The other (right of 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) following contact at elapsed times of (i) 0, (ii) 0.1, (iii) 0.2, (iv) 1.1, and (v) 5.2 days [67]. (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 LHII laden mesophase. The acceptor was mesophase without any additive. Quantitation along the length of the sample (x/L) was done spectrophometrically and is reported in normalized units of concentration (Cx/L/Cx/L-0) [64]. (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 made with 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 [78]. Reproduced from ref. 67 (a), ref. 64 (b), and ref. 78 (d). Microscale thermophoresis (MST) is a method used to monitor ligand binding [79]. 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 which change upon ligand binding. Diffusion is monitored in either a label-free mode using intrinsic

Page 11 of 28 tryptophan and/or tyrosine fluorescence or using a fluorescently labelled partner. We have shown that MST can be performed with proteins in the cubic mesophase (Figure 8) [80]. In this case, the protein was the water-soluble enzyme, lysozyme, which resides and migrates (and crystallizes) [81] 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. Since 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 membrane protein, assuming it undergoes thermophoresis.

Fig. 8. MST of lysozyme in solution and in the lipid cubic phase [80]. For the solution measurements (black traces), the lysozyme concentration used was 20 mg/mL in Milli-Q water. In meso measurements (blue traces) were made using cubic phase prepared with monoolein at 40 %(w/w) hydration using a lysozyme solution at 50 mg/mL (final lysozyme concentration in meso was 20 mg/mL). The temperature jump was activated at time 0 s and was deactivated at time 31 s. Six replicate samples were used for both sample types. Clearly, lysozyme undergoes positive thermophoresis in bulk solution and within the aqueous channels of the cubic mesophase. Reproduced from ref. 79.

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) [82]. (b) Spectrophotometry of OpcA in surfactant solution and in the cubic mesophase phase. 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 [54]. (c)

Page 12 of 28 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 [55]. Panel a is reproduced from ref. 82. Panel b is reproduced from ref. 54. Panel c is reproduced from ref. 55.

Page 13 of 28 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 [82], which plays a role in bacterial photosynthesis. It has bacterial chlorophylls and carotenoids as light gathering pigments. The absorbance spectrum of the protein in detergent solution and reconstituted into the bilayer of the cubic phase is shown in Figure 9a. They are remarkably similar with features recognizable for both pigment types consistent with a healthy protein. Similar measurements have been made with other pigment containing proteins such as bacteriorhodopsin [83], the photosynthetic reaction center [84] and visual rhodopsin [85]. In the case of bacteriorhodopsin, the light cycle has been investigated with crystals grown by the in meso method [86-88]. With rhodopsin, photoactivation and coupling with transducin was demonstrated directly in the cubic phase [85]. The UV absorbance and fluorescence characteristics of tryptophan can be used to report on the environment of that aromatic residue in a protein [89-92]. 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 (Figure 9b) [54]. 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 above for quenching by bromine in bromo-MAG, or the binding of a ligand, as will be described below. Circular dichroism is used to report on the secondary structure of a protein [93]. The useful range in which CD measurements are performed typically extends from 260 nm to 190 nm. Since the lipid used to make the cubic phase has an absorbance that is strong below about 215 nm [57], 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 provide insight into the conformation of the protein, at least in different states of dispersion, as shown for the vitamin B12 binding protein, BtuB (Figure 9c) [55]. The minimum in the CD spectrum is characteristic of β-sheet and is consistent with the known βbarrel structure of BtuB. These data support the view that the protein retains a native conformation whilst reconstituted into the bilayer of the cubic mesophase. Functional Characterization – Enzyme Activity Kinase activity – DgkA. DgkA is a kinase that functions as a homotrimer in the bacterial cytoplasmic membrane [94]. It is responsible for the ATP-dependent phosphorylation of diacylglycerol forming phosphatidic acid for use in phospholipid and membrane-derived oligosaccharide synthesis. The enzyme is promiscuous in that it can use a number of substrates other than diacylglycerols, including monoacylglycerols such as monoolein [52, 95]. The structure of DgkA was solved using crystals grown by the in meso method [10, 96, 97]. In preliminary work done whilst the crystal structure determination project was in train, DgkA was shown to be enzymatically active in the lipid cubic phase [52]. Given that the enzyme is promiscuous and that it can use monoolein as a substrate, we chose to use monoolein both as the host lipid creating the cubic mesophase membrane in which DgkA resides for crystallogenesis and as a substrate with which to monitor its kinase activity. The product of the kinase reaction with monoolein is lyso-phosphatidic acid (lysoPA) which is a potent surfactant. After prolonged activity where large quantities of monoolein are converted to lysoPA, the mesophase eventually falls apart as the surfactant dissolves the mesophase. However, in the

Page 14 of 28 early stages of the reaction, the mesophase remains intact and mechanistic enzymology studies can be performed with this sticky and viscous material. DgkA activity in the cubic phase was assayed in two ways. In the first of these, the product of the reaction, lysoPA, was monitored using thin layer chromatography (TLC) [52]. The latter method enables convenient separation of substrate from product on a TLC plate. Both can be visualized by wet ash staining. For the reaction to take place however, the ATP substrate must diffuse from the bulk bathing solution into and throughout the enzyme-laden mesophase wherein the enzyme is surrounded (and saturated4) by its lipid substrate, monoolein. That the mesophase is porous and that it should support such water-soluble nucleotide mobility was convincingly demonstrated in the ADP release studied described earlier (Figure 5). The data in Figure 10 show that indeed, DgkA was active in the mesophase as evidenced by the formation of lysoPA in a DgkA-dependent manner.

Fig. 10. TLC analysis of the substrate for and product of DgkA acting on monoolein in the cubic phase [52]. Lane 1, monoolein. Lane 2, lysoPA. Lane 3, monoolein and lysoPA. Lane 4, reaction mix, without DgkA. Lane 5, reaction mix, plus DgkA. The reaction was run overnight at 30 °C. Lanes 4 and 5 show DgkA-dependent conversion of substrate to product (Lane 5). The control (Lane 4) illustrates that non-enzymatic phosporylation of monoolein by ATP takes place at a low level. Reproduced from ref. 52. The second method used to monitor DgkA activity in situ in the cubic mesophase was by way of a coupled enzyme system [52, 98]. This is a spectrophotometric method that couples DgkA action with sequential pyruvate kinase (PK) and lactate dehydrogenase (LDH) activity. Thus, for every mole of ATP consumed and ADP produced by DgkA one mole of NADH is consumed. The loss of NADH can be conveniently monitored by tracking absorbance at 340 nm (A340) in a continuous manner using a multi-well 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 sidewall of the well out of the way of the interrogating light beam (Figure 11). The reaction was initiated by adding 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 dihexanoylglycerol (DHG) as lipid substrate and cardiolipin as activator, the reaction begins immediately without any lag (Figure 11b). However, for DgkA in the lipid mesophase a well-defined lag in the reaction progress curve is observed (Figure 11c). This is expected given that time is needed for ATP to diffuse into the 4

The concentration of monoolein in the pure cubic mesophase is about 2 molar.

Page 15 of 28 mesophase for reaction and for the product, ADP, to diffuse out for use by the coupling enzymes in the bathing aqueous solution (Figure 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 (Figure 11c). The linear region can be used for initial rate calculations and for subsequent kinetics profiling studies.

Fig. 11. Monitoring DgkA kinase activity in the cubic phase [52]. (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, c) Reaction progress in surfo and in meso at 30 °C. In (b), the reaction was in surfo with substrate, DHG, and cardiolipin, activator. In (c), the reaction was carried out with DgkA reconstituted in the mesophase formed by monoolein which doubles as substrate. The DgkA progress curve (solid line) is corrected (dashed line) for background changes recorded without enzyme (dotted line). Reproduced from ref. 52. The specific activity (SA) of the kinase in detergent solution with monoolein as substrate was 27 U/mg [52]. An operational SA of 17 U/mg was recorded for DgkA reconstituted into the monooleinbased mesophase under standard conditions (Figure 11b). Importantly, the two systems are distinctly different in that one consists of a mixed micelle, the other 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 (Figure 6) and in meso activity data (Figure 10, Figure 11c) demonstrate that DgkA kinase is active whilst 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 Mg2+-ATP as the phosphate donor. Monoolein also functions as lipid substrate. However, the concentration of monoolein in the mesophase cannot be varied easily to determine corresponding Km and Vmax values. Presumably, at 2 molar lipid in the cubic phase, monoolein saturates DgkA under conditions of assay. However, Mg2+ATP concentration can be varied and how it affected enzyme activity was quantified (Figure 12a). Michaelis-Menten kinetics were observed with Km and Vmax values of 4 mM Mg2+-ATP and 14 U/mg recorded, respectively [52]. Values of 1 mM Mg2+-ATP and 50 U/mg, respectively, were reported for DgkA in detergent solution with substrate DHG and activator cardiolipin [98]. In liposomes, the Km values range from 0.2 to 1.8 mM [97]. Thus, reconstituted in the cubic phase bilayer, DgkA functions as a well-behaved enzyme.

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Fig. 12. Dependence of DgkA activity on substrate, activator and protein concentration with the kinase reconstituted in the cubic mesophase [52]. (a) Mg2+-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) Mg2+ 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 ref. 52. DgkA in meso responded to Mg2+ concentration (Figure 12b) as it did in surfo with diolein as substrate [99]. 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 [100], had the same relative activities in meso and in surfo as the wild type kinase [52]. Given that the CLLD construct is highly stable [100], 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 upon soaking [10], whilst the second form survived the soaking and produced a structure of the complex [96]. In addition, we have demonstrated in a later study that DgkA can be refolded from acidic urea in a detergentdepleted form, to a catalytically functional form in the cubic mesophase [101]. DgkA activity rate was dependent on the concentration of enzyme in the cubic phase with rate increasing linearly initially, as expected (Figure 12c) [52]. 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 activity – PgsA. Phosphatidylglycerol phosphate (PGP) synthesizing enzyme, PgsA, plays a key role in bacterial phospholipid synthesis [102]. This small membrane enzyme converts cytidine diphosphate-diacylglycerol (CDP-DAG) and glycerol 3-phosphate (G3P) to PGP and cytidine monophosphate (CMP). PGP is subsequently converted to phosphatidylglycerol (PG) and cardiolipin (CL) [103]. PgsA, has been assayed whilst reconstituted in the lipid cubic phase (Figure 13) [52]. The lipid substrate, CDP-DAG, and product, PGP, remain in the bilayer of the mesophase as their water-soluble counterparts, G3P and CMP, respectively, move in and out of the enzyme-laden bolus (Figure 13a). Because CMP absorbs strongly at 272 nm, it is possible to quantitatively track PgsA activity in the mesophase, as was done 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.

Page 17 of 28 To monitor PgsA activity in meso, the enzyme was reconstituted into the cubic phase as described above for DgkA except that the hosting monoolein mesophase was pre-doped with substrate CDPDAG (Figure 13a). The concentration of the PgsA solution used for mesophase preparation was 0.35 mg/mL. 10 µL of the enzyme-laden mesophase was dispensed in 1-µL boluses along the side wall of a well in a multiwell plate out of the lightpath of the plate reader, as described above 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 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 Figure 13b-d 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 labelling such as with radioisotopes 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 therapeutics design and discovery.

Fig. 13. Tracking PgsA converting CDP-DAG to PGP in the lipid cubic phase [52]. (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 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,d) Dependence of enzyme activity in the cubic phase on glycerol 3-phosphate (c), and on activator, MgCl2 (d), concentration. For glycerol 3-phosphate, 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 made with 0.17 mol% of CDP-DAG in monoolein in c and d. Reproduced from ref. 52. Acyltransferase activity – PlsY. Phosphatidic acid is an essential intermediate in phospholipid biosynthesis. It is formed by two consecutive acylations of G3P. The first, catalysed by G3P acyltransferases, is known as the committed step for phospholipid synthesis [102, 103]. Two distinct G3P acyltransferases, PlsB [104, 105] and PlsY [106], exist in bacteria. Unlike the conventional type PlsB which uses thioesters (acyl-CoA or acyl-carrier protein) as the acyl donor [107], PlsY uses an unusual acyl donor, acyl-phosphate (acyl-P) [106, 108, 109]. PlsY shares neither sequence nor structure homology with other known acyltransferases, 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 [110, 111], in support of which a high-resolution structure was sought. The in meso method delivered crystals [37] and a structure [13]. During the course of the investigation, the enzyme was characterized whilst reconstituted in the bilayer of the lipid cubic phase [13] as a prelude to crystallogenesis.

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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 Pisensor, a fluorescently labelled phosphate binding protein that shows enhanced fluorescence upon ligand binding. LCP reconstituted with the enzyme PlsY and its lipid substrate, acyl-P, is dispensed on the side wall of a microplate well. The LCP also contains trace amount of Pi which need to be removed to avoid interference with the Pi-biosensor. This is conveniently done by repeatedly soaking and rinsing the sticky LCP with Pi-free buffer. To initiate the reaction, the water-soluble substrate, G3P, was added to the bathing solution. As demonstrated with ADP (Figure 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 where 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-minute 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 levelling 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 lipid product, lysoPA; lane 4, monoolein with dodecylmaltoside; lane 5, reaction mix without the enzyme; lane 6, reaction mix with the enzyme. (d, e) Michaelis–Menten and Lineweaver–Burk plot (insets) of the substrate concentration-activity relationship. One unit (U) defines the enzymatic conversion of one micromole of substrates to products per minute. (f) Effect of enzyme loading on rate of activity. The inset shows data points for the low enzyme loading region of the main plot. Results in (d–f) were from triplicate measurements. Reproduced from ref. 13. 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 Pi (Figure 14). The Pi-releasing activity of the enzyme can be monitored using a Pi-biosensor [112], whose fluorescence increases due to conformational changes upon Pi-binding. Initial attempts to assay PlsY

Page 19 of 28 in detergent micelles failed because of the presence of contaminant Pi that was either carried over from the chemical synthesis of acyl-P or produced as a result of acyl-P degradation. Thus, the 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/hydrophilicities. To this end, PlsY and acyl-P were reconstituted into the LCP which was then applied as adhering threads to the microplate wells as done for DgkA and PgsA. Pi-free buffer was added to soak and to rinse the LCP several times 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 (Figure 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 with an excitation wavelength of 445 nm and emission wavelength of 500 nm. Reaction progress curves show that the fluorescence of the Pi-biosensor increased as a function of time in a substrate and PlsY-dependant manner (Figure 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 lag phase lasts about 2 min, which is less than that observed with DgkA (compare Figures 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 Figure 14c, lysoPA was detected in the mesophase loaded with PlsY and acyl-P following 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 Vmax of 34.6 μmol min−1 mg−1 and apparent Km values of 0.1 mol% acyl-P (relative to the host LCP lipid monoolein) (Figure 14d), and 1.4 mM G3P (Figure 14e). Further, the rate scaled linearly with enzyme concentration at low concentrations consistent with PlsY behaving as a catalyst (Figure 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 crystallization leading to structure determination. Functional characterization - Ligand 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 whilst reconstituted in the mesophase prior to crystallogenesis. The vitamin B12 (cyanocobalamin, CNCbl) transporting protein, BtuB, is one such protein. It is a β-barrel protein that resides in the bacterial outer membrane [113, 114]. We used two approaches to gain insights into its ‘state of health’ in the cubic mesophase [55]. The first involved equilibrating a bolus of BtuB-laden mesophase with buffer containing CNCbl. Since CNCbl is pink, the bolus acquired a distinctly pink color while reference mesophase with no BtuB remained colorless (Figure 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 where CNCbl absorbs maximally. Binding was also determined by measuring ligand-induced quenching of intrinsic fluorescence in BtuB [55]. To this end, the protein was reconstituted in the cubic mesophase using a solution of apoBtuB in 0.1 %(w/v) LDAO. The BtuB-laden cubic phase was combined with a solution containing 0 10 µM CNCbl. Fluorescence was recorded in the 320 to 360 nm region with an excitation wavelength of 305 nm. Control measurements were made with apo-BtuB in detergent solution. Kd values were

Page 20 of 28 determined by Scatchard analysis [115]. 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 (Figure 15b). Similar Kd values have been reported for the native membrane-bound and solubilized forms of BtuB. It is apparent therefore that BtuB reconstitutes into the bilayer of the cubic phase in an active form prior to in meso crystallization. A similar analysis to that just described for BtuB has been performed with the sialic acid binding protein, OpcA, an adhesin in the bacterial outer membrane [54]. 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 µM sialic acid which is similar to the value obtained for the protein in detergent micelles (Figure 15c).

Fig. 15. Ligand binding to membrane proteins in the lipid cubic phase [55]. (a) Binding to BtuB of CNCbl (pink color). A bolus of cubic phase with (i, 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 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 µM, respectively. Reproduced from ref. 55 (a, b) and ref. 54 (c). 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 distinguish it from other membrane 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 [45]. The loaded microsyringe facilitates accurate and precise dispensing of mesophase boluses down to sub-nanoliter volumes for crystallization and functional screening [60, 63, 116] and for the delivery of continuous ‘jets’ for serial crystallography at synchrotron [117-119] and freeelectron laser X-ray facilities [120]. 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 subject to shaking or mixing as part of the assay protocol.

Page 21 of 28 The cubic phase spontaneously and macroscopically phase separates to coexist in equilibrium with bulk aqueous solution (Figure 3). This property reflects the fact that, as a liquid crystal, the cubic phase follows Gibbs Phase Rule [121]. Accordingly, when it’s water-carrying capacity is exceeded, 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 protein-laden mesophase equilibrates with a bathing precipitant solution. It is also exploited for membrane protein renaturation [101] 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 [47]. The cubic phase has been likened to a nanoporous 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 non-contacting aqueous channels that enmesh one another and that permeate the mesophase. Porosity is key to all applications of the cubic phase that include its functioning as a membrane mimetic. Thus, the ingredients of a bathing solution - water, substrates, ligands, salts, buffers, additives, etc. - can all diffuse into the interior of the bolus at rates that differ depending on activity (concentration) gradient, aqueous solubility, partitioning into and on the lipid bilayer, size, and shape. 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 reconstituted 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-focussed 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 functional activity. The cubic phase is optically isotropic. It is therefore non-birefringent. 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 a polarized light microscope. Isotropic also means that the bilayer of the cubic phase lacks sidedness. Thus, proteins reconstitute randomly orientated across the membrane. Whilst this is not a problem for crystallogenesis, since polar and apolar crystals can form regardless, it constitutes an obstacle to 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 then the cubic phase is analogous to micelles, bicelles and nanodiscs, which, in routine use, have no defined sidedness. The membrane of a unilamellar liposomes, however, has sidedness which 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 compartments, one on either side of the bilayer. Therefore, a watersoluble ligand, for example, can passage into and throughout one of the channels to bind to one of the extramembrane domains of a transmembrane 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’

Page 22 of 28 mimetics such as micelles, bicelles and nanodiscs. 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 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 aqueous solution divided equally between the two sides of the bilayer. The concentration of the lipid, monoolein, in the pure cubic phase approaches 2 molar. Therefore, a protein reconstituted to 0.1 mol% with respect to lipid is already at a concentration of 2 millimolar 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 membrane mimetics. The aqueous channels in the cubic phase are narrow and tortuous; they bend and twist (Figure 2). Thus, diffusion rates are less than 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 catalysed by enzymes reconstituted in the cubic phase (Figures 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 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 while the water-soluble product, CMP, diffused out for detection as the water-soluble substrate, G3P, diffused into the mesophase for reaction (Figure 13). A similar reaction type is carried out by PlsY, which likewise has water-soluble and lipid substrates and products (Figure 14). With PlsY, interfering inorganic phosphate 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 incorporates 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 crystallization. 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 molar in lipid (monoolein) concentration. This is an extraordinarily high lipid concentration. Indeed, in many structures obtained with in meso-grown crystals, 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 native-like. 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 so doing, they help identify natural lipid-binding sites in the case of lipid metabolizing and binding proteins. This was the case with the three enzymes in the bacterial lipoprotein post-translational modifying pathway [20, 21, 122]. It was also observed with GPCRs where functional and stabilizing cholesterol is present as part of co-complex structures obtained using 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.

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In sum, it is clear that the cubic mesophase is a generally useful system with which to investigate the biochemical 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 multi-well 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.

Acknowledgements We thank synchrotron facility scientists at the Advanced Photon Source, the 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.).

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Highlights 

The lipid cubic phase method for crystallizing membrane proteins is responsible for over 700 structure records in the Protein Data Bank.



The cubic phase functions not only in crystallogenesis but also as a biomembrane mimetic of extraordinarily high density.



The optical and rheological properties of the mesophase along with its porous bicontinuous nature make it a useful and versatile medium for investigating the biophysical and biochemical properties of membrane proteins.



Functional characterization of proteins reconstituted in the membrane of the cubic phase is possible by spectroscopic interrogation of the mesophase itself and of the aqueous solution in which it resides.



The cubic phase is compatible with an array of additives and temperatures making it an easy system to work with.



Despite its viscous and sticky nature, tools, materials and procedures are available for the facile handling and exploitation of this fascinating liquid crystalline material.