Exploring Thein Meso Crystallization Mechanism by Characterizing the Lipid Mesophase Microenvironment During the Growth of Singl

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Exploring the in meso crystallization mechanism rsta.royalsocietypublishing.org by characterizing the lipid mesophase microenvironment Research during the growth of single Cite this article: van`tHagLet al.2016 transmembrane α-helical Exploring the in meso crystallization mechanism by characterizing the lipid peptide crystals mesophase microenvironment during the 1,3,4 2,5 growth of single transmembrane α-helical Leonie van `t Hag , Konstantin Knoblich , Shane Phil.Trans.R.Soc.A peptide crystals. 374: A. Seabrook6, Nigel M. Kirby7, Stephen T. Mudie7, 20150125. http://dx.doi.org/10.1098/rsta.2015.0125 Deborah Lau4,XuLi1,3, Sally L. Gras1,3,8,Xavier 4 2,5 2,5 Accepted: 12 February 2016 Mulet , Matthew E. Call , Melissa J. Call , Calum J. Drummond4,9 and Charlotte E. Conn9 One contribution of 15 to a discussion meeting 1 issue ‘Soft interfacial materials: from Department of Chemical and Biomolecular Engineering, and 2 fundamentals to formulation’. Department of Medical Biology, The University of Melbourne, Parkville, Victoria 3052, Australia Subject Areas: 3Bio21 Molecular Science and Biotechnology Institute, physical chemistry, biochemistry The University of Melbourne, 30 Flemington Road, Parkville, Victoria 3052, Australia Keywords: 4 in meso crystallization, cubic mesophase, CSIRO Manufacturing Flagship, Private Bag 10, Clayton, Victoria local lamellar phase, DAP12. 3169, Australia 5Structural Biology Division, The Walter and Eliza Hall Institute of Authors for correspondence: Medical Research, 1G Royal Parade, Parkville, Victoria 3052, Australia 6 Calum J. Drummond CSIRO Manufacturing Flagship, 343 Royal Parade, Parkville, e-mail: [email protected] Victoria 3052, Australia Charlotte E. Conn 7Australian Synchrotron, 800 Blackburn Road, Clayton, Victoria 3168, e-mail: [email protected] Australia 8The ARC Dairy Innovation Hub, The University of Melbourne, Parkville, Victoria 3010, Australia 9School of Applied Sciences, College of Science, Engineering and Health, RMIT University, GPO Box 2476, Melbourne, Victoria 3001, Electronic supplementary material is available Australia at http://dx.doi.org/10.1098/rsta.2015.0125 or via http://rsta.royalsocietypublishing.org. CJD, 0000-0001-7340-8611

2016 The Author(s) Published by the Royal Society. All rights reserved. The proposed mechanism for in meso crystallization of transmembrane proteins suggests that 2 a protein or peptide is initially uniformly dispersed in the lipid self-assembly cubic phase but rsta.royalsocietypublishing.org that crystals grow from a local lamellar phase, which acts as a conduit between the crystal and ...... the bulk cubic phase. However, there is very limited experimental evidence for this theory. We have developed protocols to investigate the lipid mesophase microenvironment during crystal growth using standard procedures readily available in crystallography laboratories. This technique was used to characterize the microenvironment during crystal growth of the DAP12-TM peptide using synchrotron small angle X-ray scattering (SAXS) with a micro-sized X-ray beam. Crystal growth was found to occur from the gyroid cubic mesophase. For one in four crystals, a highly oriented local lamellar phase was observed, providing supporting evidence for the proposed mechanism for in meso crystallization. A new observation of this study was that we can differentiate diffraction peaks from crystals grown in meso,from Phil.Trans.R.Soc.A peaks originating from the surrounding lipid matrix, potentially opening up the possibility of high-throughput SAXS analysis of in meso grown crystals. This article is part of the themed issue ‘Soft interfacial materials: from fundamentals to formulation’. 374 :20150125 1. Introduction Membrane protein crystallography requires complex systems to drive the formation of high- quality crystals. Lipid self-assembly systems have had some success in providing crystallization media for transmembrane proteins (TMPs) via the technique of in meso crystallization [1]. The technique is particularly useful for solving the structure of membrane proteins which are important as drug targets; more than 60% of currently available pharmaceutical compounds on the market target TMPs [2,3]. The structures of approximately 200 membrane proteins and peptides have been solved to date using in meso crystallization [4–7]. However, this represents just 1% of all structures in the Protein Data Bank (PDB), while it is predicted that in most organisms membrane proteins represent 20–30% of the proteins produced [8,9]. Success rates for crystallization of TMPs are improving; almost half of all membrane protein structures solved using the in meso method have been added to the PDB in the last 3 years [7]. Recent advances involve the use of an X-ray free-electron laser set-up [10,11] for analysing in meso derived micro- /nano-crystals. However, this method is experimentally complex and not yet widely available. The mechanisms underlying the lipid–protein interactions that contribute to the formation of high-quality crystals remain poorly resolved, contributing to low overall success rates [4,6,12]. Ultimately, successful membrane protein structure determination requires an advanced understanding of the lipid microenvironment that enables crystal formation. Nevertheless, there is very limited ongoing research in this field [13–15]. Modelling studies initially suggested that suitable targets for in meso crystallization would need to include a minimum of five transmembrane helices [16]. However, this has been recently challenged by successful use of the method to solve the structures of the transmembrane peptide gramicidin, which forms a double helix or helical dimer, as well as DAP12-TM and the designed peptide Rocker, which are single transmembrane α-helices [5,17–19]. The single transmembrane α-helix DAP12 was selected to observe crystal growth in this study. DAP12 is the signalling module of immunoreceptor complexes that are involved in a broad array of biological functions. Blockade of DAP12-dependent signals forms a new therapeutic approach for the regulation of inflammatory host responses, as it is involved in chronic inflammatory diseases [20]. The structure of the transmembrane domain of DAP12 is needed to develop comprehensive mechanistic models of the multi-subunit membrane protein complexes. DAP12 is a 12 kDa signalling peptide of 113 amino acids and the transmembrane domain is formed by 24 amino acid residues within the sequence [21,22]. The structure of the helical transmembrane portion (DAP12-TM; total 33 amino acids) has been determined in several different oligomeric forms using solution NMR [21]and a b ( ) ( ) 3 rsta.royalsocietypublishing.org ...... Phil.Trans.R.Soc.A

(c)(d) 374 :20150125

Figure 1. The tetrameric DAP12-TM crystal structure at 2.14 Å resolution (PDB 4WO1) is shown in green. The orange stick representations show structured MO molecules. (a) Side view, (b)topview,(c) packing of tetrameric complexes in crystals, side view showing Type I crystal packing and (d) arrangement within single layer looking down an axis that is normal to the membraneplane.Onesymmetricunitisshowningreenandsymmetrymatesarecolouredcyanin(c,d).Thetetramericcomplex wascrystallizedfromMOwith70mgml−1 DAP12-TM,0.1 Mbis-trispropanechloride(pH6.07),19.7% w/vPEG3350and0.269 M calcium chloride [19].

recently using in meso crystallization [19]; the structure of the tetramer is shown in ﬁgure 1.The crystallographic space group of this structure is P21212 with unit cell dimensions of 50.8 Å, 43.7 Å, ◦ ◦ ◦ 50.6 Å, 90 ,90 ,90 [19]. The most commonly used lipid phases for in meso crystallization are bicontinuous cubic phases (QII)[23–26]. They consist of a lipid bilayer draped across a periodic minimal surface of zero mean curvature (ﬁgure 2 [27,28]). The three experimentally observed cubic mesophases are the diamond D P G (QII ), primitive (QII ) and gyroid (QII ) cubic phases. Cubic phases are an ideal medium for the encapsulation of soluble, peripheral and TMPs for in meso crystallization [26]. They are the only lipidic self-assembly material to contain a continuous bilayer structure over three dimensions. Cubic phases also have large surface area to volume ratios and therefore the ability to incorporate relatively large amounts of protein [26,29]. Crystals (a)((b) c) 4 rsta.royalsocietypublishing.org ......

P D Figure 2. Schematic of self-assembled lipid structures, (a) primitive cubic phase: QII ,(b) diamond cubic phase: QII ,(c) gyroid

G Phil.Trans.R.Soc.A cubic phase: QII . grown in meso have been found to be robust and of high quality and TMPs crystallized within a lipidic cubic phase are more likely to retain their activity [30]. For in meso crystallization, monoolein (MO) is the most commonly used lipid to date [5,7,31]. It has a monoglycerol head D 374 group and a singly unsaturated C18 hydrocarbon chain. It forms a robust diamond cubic QII phase in excess water and at room temperature (lattice parameter approx. 105 Å) and is readily :20150125 available at relatively low-cost [32,33]. MO cubic phases are commonly doped with 5–10% w/v cholesterol for in meso crystallization [5,34]. The proposed mechanism of membrane protein crystallization within the lipidic mesophase suggests that the protein is initially uniformly dispersed in the cubic phase but that the crystals grow from a local lamellar phase, which acts as a conduit between the crystal and the bulk cubic phase [35,36]. However, evidence for this mechanism relies on a single experimental study on one membrane protein, bacteriorhodopsin, carried out in 2007. The study, using micro-focused X-ray diffraction (XRD), showed that diffraction from the lamellar phase was only observed at the interface between the crystals and the cubic phase and not in the bulk cubic phase. The proposed mechanism is supported by indirect evidence, including the layered, Type I, crystal packing displayed by all membrane proteins crystals formed in meso [7], freeze-fracture electron microscopy (EM) studies on acetylcholine-receptor microcrystals [14] and atomic force microscopy (AFM) on bacteriorhodopsin (bR) crystals [15]. However, in the past 8 years, no additional direct experimental evidence on the highly localized cubic–lamellar transition, which is thought to occur during crystal growth, has been obtained. This may reflect the intrinsic complexity of the original experiment. A specially designed sample cell (20 μm thick) and a micro-focused (400 and 5 μm diameter) X-ray beam were used; major problems with radiation damage were encountered. In addition, analysis of the experimental results was complicated by the addition of the detergent octyl glucoside (OG) which in itself can induce lamellar phase formation [13]. Herein we describe an updated, relatively non-complex, experimental protocol to investigate the localized microenvironment around crystals grown in meso. Crucially, this should allow the proposed mechanism to be tested on numerous membrane proteins and peptides, to confirm whether the proposed mechanism is generally applicable. Standard commercially available 96-well sandwich plates (100 μm spacing) and a standard set-up for small angle X- ray scattering (SAXS) measurements were used. Multiple SAXS spectra were acquired to sample the surface of each well using a beam size of 50 × 50 μm and a step size of 25 or 37.5 μm. No detergent was used for solubilization of the peptide [18,19] which reduced the complexity of the system further. Problems with radiation damage were not encountered and very good- quality data were obtained. In this study, the local microenvironment around crystals of the single transmembrane α-helix DAP12-TM [19] was investigated using synchrotron SAXS. The experimental conditions (crystals were grown from the MO cubic phase doped with 5% w/v cholesterol) were based on those recently used to solve the structure of the DAP12-TM peptide using in meso crystallization [19]. 2. Material and methods 5 rsta.royalsocietypublishing.org (a) Materials ...... MO was obtained from Nu-Check Prep, Inc. (Waterville, MN, USA). Cholesterol and hexafluoroisopropanol (HFIP) were purchased from Sigma-Aldrich (St Louis, MO, USA). Hamilton syringes were purchased from Hamilton Company (Reno, NV, USA) and syringe couplers from TTP Labtech (Cambridge, MA, USA). The 96-well Laminex UV plastic plates and Laminex UV plastic covers were bought from Molecular Dimensions (Suffolk, UK). All crystallization chemicals with greater than 99.9% purity were sourced from Sigma-Aldrich. Lysozyme (L6876) was dissolved into a solution of 50 mM sodium chloride with 50 mM tris

chloride at pH 7.0 (again greater than 99.9% purity sourced from Sigma-Aldrich). Phil.Trans.R.Soc.A

(b) DAP12-TM peptide production and purification The disulfide-linked human DAP12-TM peptide CSTVSPGVLAGIVVGDLVLTVLIALAVYFLGRL was produced in Escherichia coli and purified following the published procedure [37]. The peptide was aliquoted and stored as a lyophilized product. 374 :20150125 (c) Plate set-up DAP12-TM was weighed and mixed with MO and 5% w/v cholesterol in a microfuge tube. A homogeneous solution was obtained by adding 500 μl of HFIP with brief vortexing and gentle heating. The sample was frozen in liquid nitrogen and then lyophilized overnight. ◦ The peptide/lipid mixture was heated to 50 C to melt the lipid and loaded into a 100 μl Hamilton syringe. Using a second syringe loaded with water and a syringe coupler, the peptide/lipid mixture was mixed until homogeneous with ultrapure water to achieve 40% w/w final water content. The peptide-containing mesophase was loaded in the 96-well sandwich crystallization plate (100 μm spacing, 0.2 μl per well) and a precipitant solution was deposited on top (1 μl per well) using the Mosquito LCP robot (TTP Labtech) based at the Collaborative ◦ Crystallization Centre (C3), CSIRO, Parkville, Australia. Plates were sealed and kept at 20 Cin a Gallery 700 incubator coupled to a Minstrel HTUV imaging system (Rigaku, The Woodlands, TX, USA) [38]. The DAP12-TM crystals were imaged using an Eclipse 80i microscope (Nikon Instruments, Melville, NY, USA) 5 days after plate set-up. DAP12-TM was crystallized at a final − concentration of 10 mg ml 1 in MO with 5% w/v cholesterol using 96 different screen conditions within this range: 0.1 M bis-tris propane chloride (pH 6.2–6.4), 19–22% w/v polyethylene glycol (PEG) 3350 and 0.03–0.07 M calcium chloride [19]. In the case of lysozyme, a similar procedure was followed but MO was loaded into one − 100 μl Hamilton syringe and lysozyme at 10 mg ml 1 in 50 mM sodium chloride with 50 mM tris chloride at pH 7.0 was loaded in the second syringe. Mixing, plate set-up and crystal growth monitoring were performed as for DAP12-TM. Lysozyme crystallized in the presence of MO under three different crystallization conditions was used: the first condition was 1.0 M sodium chloride and 0.1 M sodium acetate-acetic acid (pH 4.5). The second condition was 1.1 M sodium malonate–malonic acid (pH 7.0), 0.5% w/v jeffamine ED-2001 (pH 7.0) and 0.1 M sodium HEPES (pH 7.0). The third condition was 20% w/v PEG 6000, 0.1 M trisodium citrate-citric acid (pH 4.0), 1.0 M lithium chloride.

(d) Small angle X-ray scattering measurements Structural analysis was performed at the SAXS/WAXS beamline at the Australian Synchrotron [39]. The plates were inserted into a custom-designed, temperature-controlled plate holder for ◦ data acquisition. Data were collected at 20 C, 4 or 5 days after the DAP12-TM and lysozyme crystallization plates were prepared. Scanning of the plates and mapping within individual wells was fully automated. Data were obtained using an X-ray wavelength of 1.12713 Å (11.0 keV), − 6 beam dimensions of approximately 50 × 50 μm, a typical flux of 1 × 1010 photons s 1 and an rsta.royalsocietypublishing.org exposure time of 5.0 s. Two-dimensional diffraction images were recorded on a Pilatus 1 M ...... detector, which offers very low noise and rapid data collection over a large active area. Radial averaging of the two-dimensional images produced one-dimensional scattering profiles, with the added benefit of overcoming dead space resulting from intermodule gaps.

(e) Small angle X-ray scattering data analysis Ordered nanostructured lyotropic liquid crystalline phases with distinct diffraction patterns were studied. Many orders of diffraction were observed despite the small sample size,

allowing unambiguous identification of the cubic phase. The high-throughput analysis Phil.Trans.R.Soc.A program RapidPhaseIdent that was developed at the SAXS/WAXS beamline of the Australian Synchrotron, as described in a recent article [40], was used for lipid mesophase and lattice parameter identification along with the IDL-based AXCESS software package, developed at Imperial College London [41]. The one-dimensional diffraction patterns were normalized to the beam transmitted through the sample as measured by a detector mounted in the beamstop [39]. Peak areas were determined 374 by fitting a Gaussian with four terms within a specific q-range for each peak. This was achieved :20150125 in a high-throughput fashion using a dedicated feature within the SAXS data reduction software named ‘scatterBrain’ [42]. ScatterBrain is developed and maintained by Dr Stephen Mudie at the Australian Synchrotron. The one-dimensional diffraction patterns presented in this paper were scaled to correct for small differences in sample thickness and density observed as shifts in intensity. A simple utility was developed to calculate the mean intensity of each profile, ignoring the peak regions and in turn calculate a simple multiplicative factor to align each sample to a nominal profile. All intensities are relative (no absolute intensity calibration was performed or required). 3. Results and discussion The DAP12-TM tetrameric crystal structure, recently solved to 2.14 Å resolution, is shown in − figure 1 [19]. Crystals with Type I crystal packing were grown from MO with 70 mg ml 1 DAP12-TM, 0.1 M bis-tris propane chloride (pH 6.07), 19.7% w/v PEG 3350 and 0.269 M calcium − chloride [19]. The peptide concentration could be reduced to 10 mg ml 1, the concentration used in this study, while still enabling successful crystal growth. X-ray diffraction studies on a − significant number of crystals grown at 70 and 10 mg ml 1 DAP12-TM showed that all crystals grown in crystallization screen conditions with calcium had the same space group and unit cell. The crystallographic space group was P21212 with unit cell dimensions of 50.8 Å, 43.7 Å, 50.6 Å, ◦ ◦ ◦ 90 ,90 ,90 [19]. The microenvironment around the DAP12-TM crystals was investigated using synchrotron SAXS analysis with a micro-sized beam approximately 50 × 50 μm in size. Under excess water conditions and with no crystallization screen components, MO with 5% w/v cholesterol and final −1 D concentration of 10 mg ml DAP12-TM (0.05 mole%) formed a QII with a lattice parameter of 10.4 nm after 5 days incubation. The DAP12-TM peptide did not significantly affect the lipid mesophase formed at this concentration, since similar values are reported in the literature for MO in excess water [32,33]. The presence of 5% w/v cholesterol also had little impact on the lattice parameter, in agreement with a previous study [43]. DAP12-TM crystals (grown with 19–22% w/v PEG 3350 as precipitant, 0.03–0.07 M calcium chloride as salt additive and 0.1 M bis-tris propane chloride (pH 6.2–6.4) as buffer) were studied ◦ after 4 days incubation at 20 C. While these conditions are not identical to those used to solve the structure in figure 1, XRD analysis of the crystals grown under these conditions confirmed that they have an identical space group and unit cell parameters. In 78% of the 96 wells, crystal growth was observed within the incubation period. On average 30–200 curved rod-shaped crystals with μ alengthofaround60 m were observed per well, together with up to 10 star-shaped fractal 7 crystals with a diameter of around 60 μm. Fractal aggregates can form during nucleation [44], rsta.royalsocietypublishing.org leading to fractal crystals when followed by rapid crystal growth. The fractal crystals were easier ...... to locate because of their size and for this reason the microenvironment around fractal crystals was preferentially chosen for analysis. G The majority of wells where crystal growth was observed adopted a QII , with a typical lattice parameter of approximately 12.1 nm (73% of wells). For 24% of wells, the major phase D was the QII with a lattice parameter of around 8.6 nm, and in 3% of wells no diffraction was observed. In some cases, a minor coexisting phase of cubic or lamellar symmetry was observed. We found no significant difference in the distribution of lipid mesophases for those wells where G no crystal growth was observed. Formation of the less-hydrated QII phase, as the major phase upon the addition of a crystallization screen containing PEG 3350 at 19%–22% w/v, is consistent Phil.Trans.R.Soc.A with previous studies on the dehydrating effect of PEG on lipid mesophases [40,45–47]. Indeed D PEG 3350 at 20% w/v was found to dehydrate the QII formed by MO in excess water to the G QII by causing a reduction in water activity [40]. These data confirm that successful DAP12-TM crystal growth indeed occurred from a bulk cubic mesophase. The lipid microenvironment around the growing crystals was scanned to assess the proposed mechanism of in meso crystallization. An array of 9 × 9 data points in the X-andY-directions 374 over a total area of either 300 × 300 μm (37.5 μm step size) or 200 × 200 μm(25μmstepsize) :20150125 was scanned around DAP12-TM crystals. The microenvironment around 72 DAP12-TM crystals (in 17 areas) was analysed. Two areas without crystals were analysed as negative controls. The microenvironment around four lysozyme crystals (in three areas), also grown in meso,was analysed as a second negative control. For soluble proteins like lysozyme, crystal growth is not expected to occur from the cubic mesophase via a lamellar conduit as these proteins do not embed within the lipid bilayer. For these negative controls, an array of 7 × 7 data points in the X-and Y-directions was analysed over an area of 300 × 300 μm(50μmstepsize). An example of the 81 aligned one-dimensional diffraction patterns of a scanned area of 300 × μ G 300 m is shown in figure 3a. All patterns show a Q√II √with√ a lattice√ parameter√ √ √ of 12.5 nm√ as the major mesophase, as indicated by the observed 6, 8, 14, 16, 20, 22, 24 and 26 reflections. The reflections indicate that the structure of the cubic phase is highly homogeneous over the entire surface of the well. Additional peaks were observed in a number of plots at q- − values of 0.123 and 0.477 Å 1 as indicated in figure 3a with the blue and red arrows, respectively. − The peak at q = 0.123 Å 1 is typical of the first-order reflection of the fluid lamellar phase (Lα) of MO. We do not observe the second-order reflection from the Lα. However,√ this peak, which is of much lower intensity than the first-order reflection, is coincident with the 22 peak from the G QII phase which may preclude its observation. The lattice parameter of the putative Lα phase is 5.1 nm, typical for the Lα phase formed by MO [32,33]. The observed Lα phase reflections were single or double spots of localized intensity located ◦ 180 apart as shown by the blue arrows in figure 3b (see enlargement in inset) rather than isotropic reflections or rings. This indicates that the lamellar phase observed is highly oriented in one or two directions and is consistent with the co-location of the lipid lamellar phase at a crystal face. The localized spots were located at different positions with respect to the beamstop for different crystals, in line with the randomly oriented nature of the growing crystals. − We suggest that the peak observed at wider angle (q = 0.477 Å 1 correlating to a distance of 1.3 nm) is associated with the DAP12-TM crystal packing and refer to this as the crystal peak. This peak could be related to non-crystallographic symmetry of the DAP12-TM chains. The tetrameric structure contains four chains arranged as two dimers (figure 1), and a typical distance between chain centres is 1.3 nm. The non-crystallographic symmetry operators are shown in the electronic supplementary material, S1. The phase distribution around the growing crystals was assessed via the relative intensity of −1 three separate peaks observed at q-values√ of 0.123, 0.129 and 0.477 Å . These peaks represent G the first-order Lα phase reflection, the 6 peak from the QII phase and a putative chain packing peak from the crystal, respectively. The area under these peaks gives an indication of the amount (a) 8 10 000 ÷6 rsta.royalsocietypublishing.org ......

÷8

0.123 Å–1

intensity 1000 ÷20 ÷22 0.477 Å–1 Phil.Trans.R.Soc.A

÷ 16 ÷24 ÷26 ÷14 374 0.1 0.2 0.3 0.4 0.5 0.6 0.7 :20150125 q (Å–1)

(b)

Figure 3. SAXS diffraction patterns from a 9 × 9 array in an area of 300 × 300 μm in the DAP12-TM crystallization well a with 19.9% w/v PEG 3350, 0.065 M CaCl2, 0.1 M bis-tris propane chloride√ (pH 6.28).√ √ ( ) Eighty-one√ √ one-dimensional-diffraction√ √ √ G patterns on a double logarithmic scale. QII reflections observed are 6, 8, 14, 16, 20, 22, 24 and 26 as G −1 indicated. The QII lattice parameter was 12.5 nm. Additional reflections observed were at 0.123 and 0.477 Å as indicated by the blue and red arrows, respectively. (b) Representative two-dimensional diffraction pattern with coordinates X = 150 μm; G Y = 150 μm from the top left corner (X5Y5) in the 9 × 9array;theQII reflections are shown. Additional reflections at 0.123 −1 −1 and 0.477 Å are indicated by the blue and red arrows, respectively. The reflection of the highly orientedα L phase at 0.123 Å isshownintheenlargementintheinset.

of each phase present at different positions. These measurements provide a relative comparison of the phases present and are not entirely quantitative due to variations in sample thickness. The area under the peaks was calculated and plotted as a function of position in X and Y as shown for the example scan in ﬁgure 4a–c. The microscope image of the area under investigation (300 × 300 μm) is shown within the black box in ﬁgure 4d. The slightly larger area of 350 × 350 μm sampled by (a)(Q G peak (b) crystal peak c) La peak || 9 0 0 0 0 0 0 3.14 0.01 0.11

0.02 rsta.royalsocietypublishing.org 50 6.29 50 50 0.23 ...... 9.43 0.03 0.34 12.57 0.04 0.46 100 15.72 100 0.05 100 0.57 18.86 0.05 0.69

m) 22.01 0.06 0.80

m 150 25.15 150 0.07 150 0.92 ( Y 200 200 200 250 250 250 300 300 300 0 50 100 150 200 250 300 0 50 100 150 200 250 300 0 50 100 150 200 250 300 X (mm) X (mm) X (mm)

(d) visible crystals(e) overlay Phil.Trans.R.Soc.A 374 100 mm 100 mm :20150125

Figure 4. Peak intensity measured across the 300 × 300√μm sample area of the one-dimensional-diffraction patterns in a G q = −1 b q = −1 figure. 3 Separate maps√ are provided for ( )theQII peak ( 6, 0.129 Å ), ( ) the DAP12-TM crystal peak ( 0.477 Å ) −1 and (c)theLα peak ( 1, q = 0.123 Å ). SAXS patterns were taken after 4 days incubation. In (d), an optical image of the scanned area, taken after 5 days incubation, is shown. The area within the black box is the investigated area of 300 × 300 μm. In (e) an overlay of (a–d) is shown; image (a,b,c) are shown with increasing transparency on top of image (d).

the X-ray beam (50 × 50 μm) is also shown. In figure 4, colour maps representing the intensity of the three different phases (cubic, crystal and lamellar, figure 4a–c) are directly overlaid with an optical image of the crystals, figure 4d. This allows for easy visual correlation of the co-localization of the crystals with each phase, figure 4e. G The map of QII peak intensity, shown in green in figure 4a, is relatively homogeneous over the scanned area. The lattice parameter of this phase is also constant. This indicates that the cubic phase is highly homogeneous over the surface of the well, both in lattice parameter and relative concentration. The domains of the cubic phases analysed were smaller than 50 μm (the beam size) since powder diffraction rings (figure 3b) were observed instead of discrete Bragg reflections. DAP12-TM crystals of up to 60 μm length and diameter formed indicating that peptide must be transported from multiple domains to the growing crystals. − The maps of the crystal peak intensity in red (q = 0.477 Å 1, figure 4b)andLα peak intensity in − blue (q = 0.123 Å 1, figure 4c) are heterogeneous with localized areas of intensity. In the overlay G image in figure 4e, the intensity of QII is shown in green, the crystal peak in red and the Lα phase in blue on top of the visible crystals (figure 4d) with increasing transparency. Areas with both the G crystal peak and Lα peak show up as purple. The green colour representative of the QII is not always visible due to the other overlaying images. Figure 4e shows that the Lα phase signature was only present close to one of the DAP12-TM crystals. The intensity of the Lα peak was highest towards the edge of the crystal, suggesting that the lamellar phase is concentrated in this area. This distribution in intensity, with the lamellar phase concentrated towards the edges of the crystal, was clearly observed in additional scans of the microenvironment around growing crystals; see for example figure 6c and the electronic supplementary material, figures S2.4 and S2.8. In figure 4c, electronic supplementary material, figures S2.4(C) and S2.8(C), an arrow shows the intensity gradient. The observation is consistent with the theory of a highly oriented lipid lamellar phase conduit between the cubic phase and the crystal and provides supporting evidence for the proposed mechanism of in meso crystallization. That is, we suggest that the Type I packing DAP12-TM crystals with a lamellar spacing of 5.1 nm 10 G grew from the QII via a highly oriented lipid lamellar Lα phase with a lattice parameter of rsta.royalsocietypublishing.org 5.1 nm. This result is consistent with the previous finding that the transition from a lamellar ...... fluid mesophase to an inverse bicontinuous cubic mesophase can be a low-energy transformation process [48]. It can also be seen in figure 4e that the areas where the crystal peak was observed correlated − well with the presence of visible crystals, linking the diffraction peak at 0.477 Å 1 with the DAP12-TM crystals. In this scan, three out of four crystals observed showed the crystal peak. Two additional peaks at wider angles were consistently observed in numerous SAXS scans − at 0.587 and 0.677 Å 1 and were also found to be associated with the DAP12-TM crystals. The − three crystal peaks (q = 0.477, 0.587 and 0.677 Å 1) for the DAP12-TM crystals correlate to a distance of 1.3, 1.1 and 0.9 nm, respectively. As mentioned above, the crystal peaks could be Phil.Trans.R.Soc.A related to non-crystallographic symmetry of the DAP12-TM chains. These peaks correlate well with typical distances between chain centres, which are 1.3, 1.1 and 1.0 nm. An example of the 81 diffraction patterns of an SAXS scan with all three crystal peaks is shown in figure 5a (see inset for − enlargement of area with crystal peaks). Additional small peaks seen in the q-range 0.4–0.7 Å 1 were not consistently observed and are assumed to be noise. Analysis of the intensity of the three crystal peaks, shown in red, cyan and yellow in figure 5b, confirmed that they all correlated with 374 the two visible crystals. The screen conditions in all wells studied were very similar, so that :20150125 DAP12-TM crystals with the same space group and unit cell formed under all conditions. The − crystal peak at q = 0.477 Å 1 was most frequently observed and had the highest intensity, and this peak was therefore used for subsequent analyses. Three additional representative examples from SAXS scans around visible DAP12-TM crystals are shown in figure 6. The remaining images from all scans around DAP12-TM crystals and the results of the negative controls are provided in the electronic supplementary material, S2. A similar distribution of phases was observed for all wells studied. Again, the crystal and lamellar peaks were invariably associated with the visual observation of crystals. The lipid mesophase microenvironment was analysed around 72 DAP12-TM crystals as summarized in table 1 (see remaining images in the electronic supplementary material, S2, figures S2.1–S2.16). We note that the location of 50% of crystals was coincident with the crystal peak. Only approximately one in four crystals (24%) coincided with the lamellar peak. For 11% of DAP12-TM crystals analysed both the crystal peak and Lα peak were observed, while 38% of crystals did not show either peak. We note that for wells with no crystal growth, neither the lamellar peak nor the crystal peak was observed at any point (see the electronic supplementary material, S2, figures S2.17 and S2.18). In summary, the presence of the lamellar and crystal peaks was invariably associated with the visible observation of a crystal; the peak corresponding to the crystal phase was coincident with the observation of the crystal, whereas the lamellar peak appeared to be concentrated at the edges of the crystal. However, these peaks were not observed for every crystal. We note no correlation between the presence of the lamellar and/or crystal peaks and the rate of crystal growth. The crystal growth from 3 h until 7 days for the sample shown in figure 4,recorded by the automated Minstrel HTUV imaging system, is shown in figure 7. Crystal growth appears to have slowed down significantly after 2 days for all crystals. SAXS analysis was performed after 4 days. It is possible that some crystals may have already phase-separated from the cubic phase with the loss of the lamellar phase conduit. The highly oriented lamellar phase may also not be in the correct orientation for diffraction for all crystals. The plane of the lamellae and the X-ray beam must be parallel for diffraction to occur, as was reported for the bR crystals [13]. Finally, the absence of the lamellar peak for some crystals may reflect the low intensity of this peak. For the lysozyme scans, 75% of crystals co-localized with a crystal peak at q = 0.506, 0.705 − or 0.721 Å 1 (electronic supplementary material, S2, figure S2.19–S2.21) which correlates to a distance of 0.9–1.2 nm. For lysozyme, each originates from a different crystal packing, whereas for DAP12-TM the three crystal peaks are believed to originate from a single crystal. The lysozyme (a) ÷6 400 0.677 Å–1 11 rsta.royalsocietypublishing.org

350 ...... 10 000 0.587 Å–1 0.477 Å–1 300

intensity 250 ÷8

0.123 Å–1 200 0.40 0.45 0.50 0.55 0.60 0.65 0.70 1000 q (Å–1) Phil.Trans.R.Soc.A intensity ÷20 ÷22 ÷ 16 ÷24 ÷26 ÷14 374

0.1 0.2 0.3 0.4 0.5 0.6 0.7 :20150125 q (Å–1)

(b)

100 mm

Figure 5. (a) SAXS diffraction patterns from 9 × 9 array in an area of 300 × 300 μm in a DAP12-TM crystallization well with 20.7% w/v PEG 3350, 0.060 M CaCl2, 0.1 M bis-tris propane chloride (pH 6.38).√ Eighty-one√ √ one-dimensional-diffraction√ √ √ √ √ patterns G on a double logarithmic scale are shown. QII reflections observed are 6, 8, 14, 16, 20, 22, 24 and 26 as G −1 indicated. The QII lattice parameter was 12.3 nm. Additional reflections observed were at 0.123, 0.477, 0.587 and 0.677 Å as indicated by the blue, red, cyan and yellow arrows, respectively. The area with q of 0.4–0.7 Å−1 is enlarged in the inset. (b) Overlay of peak intensity maps representing peak areas at q of 0.477 Å−1 (red), 0.587 Å−1 (cyan) and 0.677 Å−1 (yellow) with G visible crystals. Analysis including the QII and Lα peaks is shown in the electronic supplementary material, S2 and figure S2.1.

crystals observed might have a different crystallographic space group and unit cell as they were grown with signiﬁcantly different crystallization screen conditions. All DAP12-TM crystals were grown with very similar screen conditions and had the same space group and unit cell. This result suggests that different types of peptide and protein crystals can lead to diffraction peaks at wide angles. None of the lysozyme crystals co-localized with a local lamellar phase, as expected for proteins that crystallize in the aqueous phase rather than from the lipid bilayer under conditions of in meso crystallization [49,50]. (a) 12 rsta.royalsocietypublishing.org ......

100 mm Phil.Trans.R.Soc.A

(b) 374 :20150125

100 mm

(c)

100 mm

Figure 6. Three representative peak intensity maps overlaid on crystal images as shown in figure.( 4 a) Crystallization conditions: 20.6% w/v PEG 3350, 0.061 M CaCl2, 0.1 M bis-tris propane chloride (pH 6.31). Scan area: 300 × 300 μm(b,c) Two regions from one well with crystallization conditions: 20.7% w/v PEG 3350, 0.060 M CaCl2, 0.1 M bis-tris propane chloride (pH 6.38). Scan area: 200 × 200 μm. The peak intensity maps and optical images are shown individually in the electronic supplementary material, S2, figures S2.2–S2.4.

Crystal peaks were not observed for all DAP12-TM and lysozyme crystals. As for the Lα phase, the crystals may not always be in the correct orientation to result in crystal peak diffraction at wide angles. The absence of the crystal peaks for some crystals may also reﬂect the low intensity of these peaks. (a) 3 h (b) 1 day 13 rsta.royalsocietypublishing.org ......

100 mm 100 mm

(c)(2 daysd) 7 days Phil.Trans.R.Soc.A 374 :20150125 100 mm 100 mm

Figure7. OpticalimagesofDAP12-TMcrystalgrowthinanareaof350 × 350 μmafter(a)3h,(b)1day,(c)2daysand(d)7days. ImagesshownherearetakenwiththeMinstrelHTUVimagingsystem[38].ThesecrystalswereusedforSAXSanalysis,asshown in figures 3 and 4.

Table 1. Data are provided for the total number of crystals analysed, the number of crystals that are co-localized with a crystal peak (crystal peak), the number of crystals that are co-localized with a lamellar peak (Lα peak), the number of crystals that are co-localized with both a lamellar and a crystal peak (crystal + Lα peak) and the number of crystals where neither a lamellar nor a crystal peak was observed (no Crystal or Lα peak). Data are presented as a summary of all crystals observed, and for two control areas where no crystals were observed. Data are also provided for the growth of four lysozyme crystals. In addition, we have provided a summary of data for the four images shown in figures 4 and 6a–c, respectively. Crystals with more than 50% of the area of the crystal present in the region sampled by the X-ray beam were counted. A table with the numbers for all individual scans is shown in the electronic supplementary material, S3 based on the images in the manuscript and electronic supplementary material, S2.

crystal + Lα no crystal or crystals crystal peak Lα peak peak Lα peak peptide/protein and corresponding image # # % # % # % # % DAP12-TM (all crystals) 72 36 50 17 24 8 11 27 38 ...... DAP12-TM(control,nocrystals)0 0 0 0000 00 ...... lysozyme(allcrystals) 4 3750000 125 ...... DAP12-TM, figure 4 4375125125125 ...... DAP12-TM, figurea 6 623311700350 ...... DAP12-TM, figureb 6 3 1330000 267 ...... DAP12-TM, figurec 6 2210015015000 ......

The highly oriented local Lα phase of MO with lattice parameter of around 5.1 nm that was observed at the edge of 24% of DAP12-TM crystals provides support for the proposed mechanism for in meso crystallization [13,35]. The crystal packing in the DAP12-TM crystals also showed a spacing of 5.1 nm between layers [19], suggesting the crystals could grow via the lipid Lα phase 14 conduit. This represents the ﬁrst direct comparison of the spacing between layers in Type I crystals rsta.royalsocietypublishing.org with the lattice parameter of the lipid Lα phase conduit. A local Lα phase with similar lattice ...... parameters was observed around bacteriorhodopsin crystals that were grown from the MO cubic phase [13].

4. Conclusion Crystallization of the transmembrane DAP12-TM peptide was found to be consistent with crystal growth occurring from the gyroid cubic mesophase via a highly oriented local lipid lamellar phase. The lamellar spacing of the Type I DAP12-TM crystals matched remarkably well with the lattice parameter of the lipid lamellar phase observed. In the negative controls, the local lamellar Phil.Trans.R.Soc.A phase was not observed. A direct observation of the local lamellar phase using SAXS has only previously been reported for a single TMP (bacteriorhodopsin). The observation of a localized lamellar phase reported here for the transmembrane peptide DAP12-TM crystals provides important additional experimental evidence as to the generality of the proposed mechanism. An improved understanding of this promising in meso crystallization technique might lead to a more 374 rational design of crystallization experiments and improved success rates. The results presented :20150125 indicated that peptide from multiple cubic phase domains was transported to the growing crystals. A new observation of this study was that DAP12-TM and lysozyme crystals can give rise to diffraction peaks at wide angles. This is of potential use in locating peptide or protein crystals in the lipidic cubic phase as they are generally hard to observe. The experiment presented here used commercially available 96-well crystallization plates and a standard set-up for synchrotron SAXS measurements which will enable further testing of the proposed mechanism on a variety of TMPs and peptides as it can readily be adapted by other research groups. Additionally, the DAP12-TM peptide was co-lyophilized with MO and cholesterol and no detergent was used for solubilization which reduced the complexity of the system further.

Data accessibility. SAXS data and original microscope images: Dryad (http://dx.doi.org/10.5061/dryad.1mr70). Authors’ contributions. L.vtH. set-up and monitored DAP12-TM crystallization, performed synchrotron SAXS measurements and data analysis and wrote the paper; K.K. produced the DAP12-TM peptide and developed reproducible crystallization protocols; S.A.S. contributed to developing crystallization protocols; N.M.K. developed and facilitated the SAXS set-up with micro-sized beam; S.T.M. developed high-throughput SAXS data analysis software; D.L. suggested and assisted with image analysis protocols; X.L. and X.M. assisted with synchrotron SAXS measurements; X.M. took microscope images of the crystals. S.L.G., M.E.C., M.J.C. and C.J.D. provided critical commentary on the results and manuscript; C.E.C. assisted with design of the study, SAXS data analysis and drafting the manuscript. All authors contributed to editing the paper and gave ﬁnal approval for publication. Competing interests. The authors declare no competing interests. Funding. This work was supported by grants from the National Health and Medical Research Council (NHMRC) of Australia (GNT1011352 to M.J.C. and M.E.C.; Independent Research Institutes Infrastructure Support Scheme (IRIISS) to the Walter and Eliza Hall Institute of Medical Research (WEHI)) and the Victorian Government (VESKI Innovation Fellowship VIF12 to M.E.C.; Operational Infrastructure Support to WEHI). M.E.C. is supported by a QEII Fellowship (DP110104369) from the Australian Research Council (ARC). M.J.C. is supported by an ARC Future Fellowship (FT120100145). S.L.G. is supported by the ARC Dairy Innovation Hub IH120100005. Acknowledgements. We thank Dr Adrian Hawley for his assistance with SAXS experiments, and we acknowledge use of the SAXS/WAXS beamline at the Australian Synchrotron. We thank Dr Janet Newman for help with robotic set-up of plates at the C3 Collaborative Crystallisation Centre, CSIRO, Parkville, Australia.

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