Effect of Lipid-Based Nanostructure on Protein Encapsulation within

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Effect of Lipid-Based Nanostructure on Protein Encapsulation within the Membrane Bilayer Mimetic Lipidic Cubic Phase using Transmembrane and Lipo-Proteins from the Beta-Barrel Assembly Machinery Leonie van 't Hag, Hsin-Hui Shen, Tsung-Wu Lin, Sally L. Gras, Calum J. Drummond, and Charlotte E Conn Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b01800 • Publication Date (Web): 21 Jun 2016 Downloaded from http://pubs.acs.org on June 24, 2016

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Effect of Lipid-Based Nanostructure on Protein Encapsulation within the Membrane Bilayer Mimetic Lipidic Cubic Phase using Transmembrane and Lipo-Proteins from the Beta-Barrel Assembly Machinery Leonie van ‘t Hag,I,II,III Hsin-Hui Shen,IV,V, Tsung-Wu Lin,VI Sally L. Gras,I,II,VII Calum J. Drummond,III,VIII,* and Charlotte E. Conn.VIII,** I

II

VII

Department of Chemical and Biomolecular Engineering, Bio21 Molecular Science and Biotechnology Institute, The ARC Dairy

Innovation Hub, The University of Melbourne, Parkville, Victoria 3010, Australia III

CSIRO Manufacturing, Clayton, Victoria 3168, Australia

IV

V

Department of Microbiology, Faculty of Engineering, Monash University, Melbourne, Victoria 3800, Australia

VI

Department of Chemistry, Tunghai University, Taichung City 40704, Taiwan

VIII

School of Science, College of Science, Engineering and Health, RMIT University, Melbourne, Victoria 3001, Australia

Corresponding Authors E-mail: *[email protected], **[email protected]

ABSTRACT A fundamental understanding of the effect of amphiphilic protein encapsulation on the nanostructure of the bicontinuous cubic phase is crucial to progressing biomedical and biological applications of these hybrid protein-lipid materials, including as drug delivery vehicles, as biosensors, biofuel cells and for in meso crystallization. The relationship between the lipid nanomaterial and the encapsulated protein, however, remains poorly understood. In this study, we investigated the effect of incorporating the five transmembrane and lipo-proteins which make up the β-barrel assembly machinery from Gram-negative bacteria within a series of bicontinuous cubic phases. The transmembrane β-barrel BamA caused an increase in lattice parameter of the cubic phase upon encapsulation. In contrast, the mainly hydrophilic lipoproteins BamB-E caused the cubic phase lattice parameters to decrease, despite their large size relative to the diameter of the cubic phase water channels. Analysis of the primary amino acid sequence 1 ACS Paragon Plus Environment

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was used to rationalize this effect, based on specific interactions between aromatic amino acids within the proteins and the polar-apolar interface. Other factors that were found to have an effect were lateral bilayer pressure and rigidity within the lipid bilayer, water channel diameter and size and structure of the lipoproteins. The data presented suggest that hydrophilic bioactive molecules can be selectively encapsulated within the cubic phase by using a lipid anchor or aromatic amino acids, for drug delivery or biosensing applications. INTRODUCTION Membrane proteins that are fully or partially integrated into the cell membrane serve essential cell functions assisting with signaling, adhesion, replication and nutrient import.1,2 Self-assembled lipid bilayer systems can be exploited to study these membrane or lipo-proteins in vitro.3 One such model system for membrane protein incorporation is the lipidic bicontinuous cubic phase. These cubic phases have been observed in vivo in the nuclear and mitonchondrial membranes of cells and in the endoplasmatic reticulum.4 The cubic phases observed experimentally in vitro are the gyroid (QIIG), diamond (QIID) and primitive (QIIP) cubic phases, shown in Figure 1. These phases consist of a continuous structure of surfactant bilayers and water channels, with a high surface area to volume ratio.5 For this reason, cubic phases are very useful for the encapsulation of amphiphilic transmembrane proteins for study or potential delivery of these proteins. The hydrophobic domain of an amphiphilic protein, consisting of an α-helical or β-barrel fold, can be encapsulated within the lipid bilayer and the hydrophilic domain(s), present in the extracellular fluid or cytoplasm of the cells, in the water channels.6,7 The flexible cubic phase nanostructure can also accommodate soluble and peripheral proteins with a range of molecular weights.8,9 The applications of encapsulated proteins in cubic phases include the in meso crystallization of membrane proteins,10 as well as use as biosensors11 or biofuel

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cells12 and as drug delivery vehicles for hydrophobic and amphiphilic therapeutics and medical imaging agents.13

P

D

G

Figure 1. Schematic presentation of the (A) Primitive cubic QII phase, (B) Diamond cubic QII phase, (C) Gyroid cubic QII phase. Sheets represent the lipid bilayers. Reproduced with permission from reference 14.

The nanostructure of cubic phases can be significantly impacted by the incorporation of amphiphilic proteins and peptides, and can induce phase transitions to non-cubic phases.8 The retention of the cubic phase is essential, however, for current and prospective applications of such systems. In drug delivery applications for example, the drug release rate is strongly linked to the lipid-based nanostructure.15,16 To date, we have been unable to predict phase transitions caused by the incorporation of amphiphilic proteins and peptides in the bicontinuous cubic phase.8,17-21 Additionally, very limited research has been published on the effect of incorporating lipo- or peripheral membrane proteins in the cubic phase, with most studies focusing on the same lipoprotein: cytochrome c from eukaryotes.8,22 Here we investigate a series of transmembrane and lipoproteins, which make up the β-barrel assembly machinery (BAM) complex within the bicontinuous cubic phase. The BAM complex from Gram-negative bacteria is required for newly synthesized β-barrel membrane proteins to be integrated into the outer membrane. The secreted proteins are known to contribute to bacterial virulence and are responsible for infectious diseases such as diarrhoea, whooping cough, cholera, chlamydia, and bacterial meningitis, by a range of different Gram-negative bacteria. Knowledge of the structure of the BAM complex can assist in our understanding of the mechanisms by which β-barrel membrane proteins are assembled by the BAM complex. This may help in 3 ACS Paragon Plus Environment

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understanding how to stop the assembly of virulent proteins on the cell surface and subsequently in the prevention of infectious diseases. The BAM complex consists of one integral outer membrane βbarrel protein that spans the lipid bilayer (BamA) and four peripheral lipoproteins that are anchored to the inner phospholipid leaflet of the outer membrane (BamB-E).1,23 The lipoproteins are anchored to the membrane by a covalently bound lipid via an amino-terminal cysteine. The lipid anchor is a thioetherlinked diacylglyceryl group and it contains a palmitoyl chain at the amino-terminus,24 as shown in Figure 2(A). BamA is the core subunit of the BAM complex, it contains five polypeptide transport associated (PORTA) domains in addition to the integral β-barrel as shown in Figure 3(A).25 Each PORTA domain is organized as a three-stranded β-sheet with two anti-parallel α-helices. The hydrophobic thickness of BamA is greatly reduced along the last β-strand, resulting in a marked decrease in lipid order and membrane thickness.1 This suggests that the lipid-exposed surface is less than ~27 Å, the typical value for β-barrels in the outer membrane of gram-negative bacteria.26,27 BamB forms a ring-like structure, consisting of an eight-bladed β-propellor, Figure 3(B).28 It interacts with BamA through PORTA3-4 and is the component that captures the substrate. BamC is a mixture of α-helices and β-sheets, as shown in Figure 3(C); this structure also interacts with the complex via BamD.29 BamD is composed of α-helices, as shown in Figure 3(D); interacting with BamA via PORTA5. BamD is the only essential lipoprotein of the BAM complex.30 BamE consists of three-stranded anti-parallel β-sheets packed against two α-helices, as shown in Figure 3(E), and it also interacts with the complex through BamD.31 For the lipoproteins BamBE used in this study, the lipid anchor was not present due to ease of expression. In vivo the lipid anchor is attached after translation by a number of enzymes.32


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Figure 2. Chemical structures of (A) the lipid anchor of BamB-E, and the cubic phase forming lipids (B) monovaccenin, (C) monopalmitolein, (D) monoolein, (E) phytantriol, (F) phytanoyl monoethanolamide.

Figure 3. Illustration of dimensions and secondary structures of (A) the transmembrane β-barrel BamA, ~87 kDa, pI 8.9 (PDB ID 33

33

4K3B) , the five PORTA domains are numbered from the bottom towards the β-barrel as labelled with P1-P5. The lipoproteins 28

(B) BamB, ~40 kDa, pI 4.6 (PDB ID 3Q54) , (C) BamC, ~35 kDa, pI 5.1: structure shown is from the N-terminal 21 kDa (PDB ID 29

30

3TGO) . (D) BamD, ~26 kDa, pI 5.5 (PDB ID 3Q5M) , and (E) BamE, ~11 kDa, pI 6.9 (PDB ID 2KXX).

31

β-sheets are shown in

yellow, α-helices in red, loops in green. The scale bar indicates 25 Å. The iso-electric point, or pH where the peptide exists at a 34

net charge neutral state, was calculated using the ExPASy Proteomics service using default values for resolution.


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We chose five cubic-phase forming lipids to investigate the effect of lipid chain length and branching, as well as the water channel diameter, on the encapsulation of the BamA-E. Three of these lipids are monoacylglycerol (MAG) lipids, with unsaturated alkyl chains of different lengths: monoolein (MO, 7.7 MAG), monopalmitolein (MP, 9.7 MAG) and monovaccenin (MV, 11.7 MAG). The numbers indicate the number of carbons before and after the unsaturation in the chain. The lipids selected with branched alkyl chains are phytantriol (PT) and phytanoyl monoethanolamide (PE). Molecular models of all five lipids used in this study are shown in Figure 2(B-F). The range of lipid lengths for the diamond cubic QIID phase at room temperature and in excess water is 13 – 18 Å, while the corresponding water channel diameters vary from 23 – 65 Å, as shown in Table 1. The lateral bilayer pressure profile of the selected lipids should also be considered in addition to lipid length and water channel diameter. This pressure is significantly higher in the chain region (by approximately a factor of two) for the branched alkyl chains compared to the MAGs.35 The rigidity of the lipid bilayer can be increased by the addition of cholesterol, so the MO cubic phase was studied in the absence and presence of 8% w/w cholesterol to obtain a greater systematic insight into the effect of membrane rigidity. The monoolein cubic phase is commonly doped with cholesterol for in meso crystallization, making this combination also relevant for potential applications.10 Small-Angle X-Ray Scattering (SAXS) is used to investigate the effect of the transmembrane protein BamA and the four lipoproteins BamB-E on the bicontinuous cubic mesophases formed by MV, MP, MO (+8% w/w cholesterol), PT and PE. The 30 combinations applied in this study are expected to increase our understanding of the impact of transmembrane and lipo-proteins with varying secondary structure on cubic phases with different lipid chain lengths and chain branching. The dataset also extends insights into the differences between proteins rich in α-helical or β-sheet secondary structures and their effect on cubic phase nanostructure.


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EXPERIMENTAL SECTION Materials. MO, Cholesterol, Tris, NaCl, imidazole and DDM were purchased from Sigma Aldrich (St. Louis, MO, USA). MP and MV were obtained from Nu-Check Prep, Inc. (Waterville, MN, USA). 3,7,11,15tetramethyl-1,2,3-hexadecanetriol (PT) (96%) was purchased from DSM Nutritional Products (Germany) and PE was synthesized in our laboratory.36 Ethanol and chloroform were bought from Merck & Co., Inc (Whitehouse Station, NJ, USA). Elugent detergent was purchased from VWR International, Pty Ltd (Murarrie, QLD, Australia). SD-2 96-well crystallization plates were obtained from IDEX corp (Rohnert Park, CA, USA). Expression and Purification of C-His8-BamA The medium used for protein expression was Lysogeny Broth with ampicilline (100 μg mL-1). BamA was over-produced in BL21(DE3)* cells by growing strain carrying pET 22b at 30 oC to an OD600 of 0.6 – 0.7. The growth temperature was then switched to 16 oC. Expression was induced by 0.1% arabinose overnight. Cells from the two strains were harvested by centrifuging at 8,000 g for 10 minutes at 4 oC (VWR International-102C). The pellet was resuspended in lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl and PMSF) and a protease inhibitor cocktail was added before disrupting the cells in an Avestin cell disruptor. Unbroken cells were removed by centrifuging at 26,000 g for 15 minutes. The supernatants were ultracentrifuged at 142,000 g at 4 oC for 60 min to pellet the whole membrane. The membrane pellets were resuspended in 20 mM Tris, 150 mM NaCl, 5% Elugent, pH 7.5 and were incubated overnight at 4 oC to extract the membrane proteins. The supernatants were applied to 1 mL of Ni-NTA affinity columns after adding 5 mM imidazole and were passed through the column using pump overnight at cold room. The columns were then washed with 10 mL washing buffer (20 mM Tris, 150 mM NaCl, 20 mM imidazole, 0.5% Elugent, pH 7.5). For elution 20 mM Tris, 150 mM NaCl, 500 mM imidazole, 0.5% Elugent, pH 7.5 was used. Proteins were subjected to size exclusion chromatography 7 ACS Paragon Plus Environment

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(GE healthcare, Little Chalfont, UK) in 20 mM Tris, 150 mM NaCl and 0.5% Elugent to remove aggregated proteins. Proteins were then dialysed with 20 mM Tris, 150 mM NaCl and 0.03% w/v DDM buffer solution. Expression and Purification of BamB, BamC BamD and BamE Cultures of BL21(DE3) cells carrying pET16 were grown and induced in 0.2 mM IPTG for 2 hours at 37 oC. The cells were harvested and lysed as described above without the addition of the detergents Elugent and DDM. After centrifugation of the unbroken cells and whole membrane, the supernatant was applied to 1 mL Ni-NTA resin per litre culture and was purified followed the standard protocol as previously described.37 Note that the buffers did not contain detergents. Plate Setup. A high-throughput method was used to prepare samples for Small-Angle X-Ray Scattering in standard 96-well SD-2 crystallization plates.38 MV, MP, MO, PT and PE solutions in ethanol were prepared at 200 mg mL-1. For MO with cholesterol MO was initially mixed with 8% w/w cholesterol and subsequently dissolved at 200 mg mL-1 in ethanol with a small amount of chloroform. A volume of 1.05 μL lipid solution in ethanol (0.21 mg of lipid) was dispensed into the top and bottom subwells of the plates using a Mosquito robot (TTP Labtech) based at the C3 collaborative Crystallization Centre, CSIRO, Parkville, Australia. The plates were dried under vacuum for six days to remove solvent. 20 μL of 20 mM Tris, 150 mM NaCl, 0.03% w/v DDM for BamA or 20 mM Tris with 150 mM NaCl for BamB-E was then added into the reservoirs of the wells. Using the Mosquito robot, a 0.4 µL aliquot of protein solution at 0 – 2 mg mL-1 in buffer or buffer with detergent was dispensed on the lipid film; the samples were in 66% w/w (excess) water. Protein concentrations were randomized in the rows (B-G) of the plate to eliminate deposition effects. Small-Angle X-ray Scattering (SAXS) Measurements. The lipid mesophase structure was determined in situ at 25 oC within the 96-well plates 24 hours after addition of the protein solution. The plates were 8 ACS Paragon Plus Environment

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mounted directly onto the SAXS/WAXS beamline at the Australian Synchrotron39 using a customdesigned plate holder. Position variables based on the well positions were used for automated scans across the plates. The experiments used a beam of wavelength λ = 1.23984 Å (10 keV) with dimensions of approximately 200 x 300 μm, a typical flux of 1 x 1013 photons s-1 and an exposure time of 0.5 s. 2D diffraction images were recorded on a Pilatus 1M detector, allowing rapid data collection and offering very low noise. 1D scattering profiles were obtained by radial averaging of the 2D images obtained. SAXS data analysis. Diamond cubic QIID mesophases, which are ordered nanostructured lyotropic liquid crystalline phases, with distinct diffraction patterns were investigated. Provided an adequate number of reflections are observed the diffraction patterns may be used as unambiguous identification of the phase. Representative 1D diffraction patterns obtained for diamond cubic QIID mesophases of MV, MP, MO, PT and PE with buffer or buffer with DDM, and with BamA and BamD at 1.6 – 2 mg mL-1 are shown in Figure 4. Six orders of diffraction were observed for most samples despite their small size. The highthroughput analysis program RapidPhaseIdent that was developed at the SAXS/WAXS beamline of the Australian Synchrotron was used for identification of lipid mesophases and lattice parameters.40 RESULTS AND DISCUSSION Bicontinuous Cubic Mesophases of MV, MP, MO, PT and PE with Buffer and Detergent. The transmembrane protein BamA and the lipoproteins BamB-E were incorporated within the bicontinuous cubic phase formed by six different lipid systems: MV, MP, MO, MO with 8% w/w cholesterol, PT and PE, at room temperature and in excess water. Small angle X-ray scattering was used for the characterization of the lipid mesophases formed and representative 1D diffraction patterns for the diamond cubic QIID phases are shown in Figure 4. Diffraction patterns for the lipids with buffer (20 mM Tris, 150 mM NaCl) or buffer with detergent (20 mM Tris, 150 mM NaCl, 0.03% w/v or 0.59 mM


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DDM) are shown. In addition, diffraction patterns are presented for the BamA and BamD proteins incorporated at a high concentration: 1.6 – 2.0 mg mL-1 within the cubic phase.

D

Figure 4. 1D diffraction plots of intensity on a logarithmic scale vs. q for the diamond cubic QII phase. The typical Bragg -1

-1

reflections for this phase are visible: √2, √3, √4, √6, √8 and √9. (A) MV with buﬀer, 2.0 mg mL BamA, 2.0 mg mL BamD. (B) MP -1

-1

-1

-1

with buffer, 1.6 mg mL BamA, 2.0 mg mL BamD. (C) MO with buffer and detergent, 1.6 mg mL BamA, 1.6 mg mL BamD. (D) -1

-1

-1

-1

PT with buffer, 2.0 mg mL BamA, 2.0 mg mL BamD. (E) PE with buffer, 1.8 mg mL BamA, 2.0 mg mL BamD. Lattice parameters are shown in Supplementary Information Tables S1.1 – S1.5.

All samples of the 30 different combinations of lipid and protein samples were run in duplicate; in the top and bottom subwells within the 96-well plate. As described in previous work, reproducibility between the subwells must be confirmed.19,21,38 Variation of the lattice parameters between the top and bottom subwells is shown in plots in the Supplementary Information Figures S1.1 – S1.5. All mesophases and their associated lattice parameters, as identified from the SAXS data are provided in the 10 ACS Paragon Plus Environment

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Supplementary Information Tables S2.1 – S2.5. In ~89% of wells the data were found to be reproducible, with the same mesophase present in both subwells and with lattice parameters less than 3 Å different. Irreproducible samples were not considered in further analysis. Of the five BAM proteins studied, BamA and BamD had the most significant effect on the diamond cubic QIID phase lattice parameters. This effect will be discussed in more detail in the following subsections. Even at the highest protein concentrations studied, the differences in the 1D diffraction patterns upon protein incorporation is small, as shown in Figure 4. However, due to the high reproducibility between the top and bottom subwells and the highresolution SAXS measurements performed at the Australian synchrotron we can effectively characterize the trends in lattice parameter upon protein incorporation. Significant and reproducible trends in lattice parameter were observed despite the small sample size. BamA was solubilized in buffer with detergent to enable solubilization of the hydrophobic transmembrane domain and the hydrophilic BamB-E proteins were solubilized in buffer without detergent. For this reason, the lattice parameter (lp) of the diamond cubic QIID phase with buffer and detergent, with buffer alone and water under excess hydration was investigated for all six lipid systems providing suitable background for comparison, Table 1. The lattice parameters that were observed for all lipids in excess water are consistent with values obtained in the literature from phase diagrams;36,41-44 ranging from 63 Å for PE to 128 Å for MV, Table 1. Values for MO with 8% w/w cholesterol were 2 Å higher than without cholesterol, also consistent with the literature.45,46 Values for all six lipids systems in the presence of the buffer consisting of 20 mM Tris and 150 mM NaCl were ~1 Å lower than or equal to values with water. A slight reduction in lattice parameter in the presence of 150 mM of a kosmotropic salt, like NaCl, can be explained by a reduction in water activity.40,47 Lattice parameters of QIID in the presence of buffer and the detergent DDM at 0.03% w/v were always 1 – 3 Å higher than with water and 1 – 4 Å higher than with buffer. An increase in QIID lattice parameters was previously reported when DDM was added in increasing concentration to the MO cubic phase.48 11 ACS Paragon Plus Environment

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The bilayer thickness (BL) and water channel diameters (Dw) were calculated for MV, MP, MO, PT and PE, based on the diamond cubic QIID phase lattice parameters with water, Table 1. The method, based on the work of Caffrey et al, is provided in Supplementary Information 3.49,50 These values can be used to assess the effect of lipid chain length and cubic phase nanostructure on BamA-E protein encapsulation. MV has the largest bilayer thickness and water channel diameter of the lipid systems studied. MP and MO have very similar bilayer thicknesses but MP has significantly larger water channels than MO. The branched chain lipids PT and PE have much smaller water channels than the three MAG lipids and they also have thinner bilayers, with the smallest bilayer thickness calculated for PE. The branched chains of PT and PE, Figure 2, additionally allow us to investigate the effect of chain architecture, as branched chains are known have a much higher lateral bilayer pressure in the chain region.35 The bilayer thickness and water channel diameter of MO with 8% w/w cholesterol is expected to be similar to values of MO but the rigidity of the lipid bilayer in this system is expected to be increased. D

Table 1. Experimentally obtained values of lattice parameters (lp) of the diamond cubic QII phase (Å) at 25 °C and in excess hydration (66% w/w). Results are shown with water (Lp water), buffer consisting of 70 mM Tris and 200 mM NaCl (Lp buffer) or -1

buffer with the detergent DDM (Lp buffer det). Averages of the 0 mg mL BAM protein values are shown and standard deviations were ~0.6 Å (see Supplementary Information S2). The bilayer thickness (BL) and water channel diameters (Dw) were calculated for MV, MP, MO, PT and PE.

49,50

Values for MO with 8% w/w cholesterol are expected to be similar to MO. The water

contents used in the calculations were the excess water point for each lipid, as obtained from the appropriate phase diagram, 43

49

44

41

36

-3

and were 52% , 51% , 47% , 28% and 30% respectively. The lipid densities used in the calculations were 0.942 g cm for -3

-3

MV and MO, 0.982 g cm for MP and 0.940 g cm for PT and PE.

Lipid Monovaccenin (MV) Monopalmitolein (MP) Monoolein+ 8% w/w cholesterol Monoolein (MO) Phytantriol (PT) Phytanoyl monoethanolamide (PE)

Lp buffer det. (Å) 129 119 109 108 70 64

Lp buffer (Å) 127 115 108 106 68 63


Lp water (Å) 128 116 108 106 69 63

BL (Å) 35.5 31.4

Dw (Å) 64.5 59.2

32.4 29.4 26.0

50.4 24.5 23.3

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Incorporation of the Transmembrane β-barrel BamA. The increase in lattice parameter of the diamond cubic QIID phase upon incorporation of the transmembrane β-barrel BamA is shown in Figure 5(A-B). The effect of cholesterol on the trends in lattice parameters will be discussed separately. A linear fit through the data points is shown as a guide for the eye. The incorporation of BamA into the cubic phase of all five lipids resulted in a linear increase in lattice parameter with increasing protein concentration, no plateau was observed at the highest BamA concentration studied: 2 mg mL-1. No coexisting or other mesophases were observed. The maximum increase in lattice parameter was 6 – 8% for all lipids: MV, MP, MO, PT and PE (details provided in Supplementary Information Table S2.1). The lipid-exposed surface of BamA is less than ~27 Å, based on its crystal structure in Figure 3(A) and the typical value for β-barrels in the outer membrane of gram-negative bacteria.26,27 Previous research showed that hydrophobic mismatch of a membrane protein with the lipid bilayer thickness is important in determining the response of the lipid mesophase.19,51,52 Negative hydrophobic mismatch of the hydrophobic domain with the lipid bilayers of MV, MP and MO is expected (BL 31 – 36 Å). For the branched chain lipids PT and PE, approximate hydrophobic matching is expected with a bilayer thickness of 26 – 29 Å. However, for all lipids a similar increase in lattice parameter with increasing BamA concentration was observed. This suggests that the hydrophilic PORTA domain was also important in determining the changes in nanostructure observed. No differences were observed based on lipid chain length or lipid bilayer thickness and branching.


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Figure 5. Incorporation of BamA-E into QII of (A, C, E, G, I) MV (squares), MP (diamonds), MO (triangles) and (B, D, F, H, J) phytantriol (stars) and phytanoyl monoethanolamide (crosses). Results are shown for (A-B) BamA, (C-D) BamB, (E-F) BamC, (G-


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H) BamD, (I-J) BamE. The average of top and bottom subwells is plotted (see Supplementary Information Tables in S2). A linear fit through the data points is shown as a guide for the eye. BamA was added in 20 mM Tris, 200 mM NaCl and DDM and BamB-E -1

were added in 20 mM Tris with 200 mM NaCl. A circle around data points at 2 mg mL in (C) indicate there was a coexisting P

primitive cubic QII phase with a lattice parameter of 161 Å for MV, 147 Å for MP, 137 Å for MO.

The hydrophilic PORTA domain of BamA is ~73 Å long and ~58 Å in diameter. This is slightly larger than the water channels of the cubic phases formed by the MAG lipids (Dw 50 – 65 Å) and significantly larger than the water channels of the cubic phases of the branched chain lipids (Dw 23 – 25 Å). This geometrical mismatch is expected to be an important factor that causes the swelling of the QIID phase nanostructure, as was observed upon BamA incorporation. When the hydrophilic domain size is much larger than the water channel diameter, slight regions of disorder might be formed.53 The full width at half-maximum (FWHM) was calculated for the most intense peak for each phase. No signs of peak broadening were observed suggesting that there was no significant increase in disorder throughout the sample and that any regions of disorder must be highly localized. These results suggest that the flexible cubic phase nanostructure is able to accommodate amphiphilic proteins of a range of sizes. These results are consistent with previous studies on amphiphilic β-barrel and α-helical transmembrane proteins, where an increase in cubic phase lattice parameter, or swelling of the cubic phase nanostructure,

was

observed

upon

protein

encapsulation.8,18-20,54

The

exceptions

were

bacteriorhodopsin18 and intimin (unpublished work), mostly hydrophobic transmembrane proteins, in which case no changes in lattice parameter were observed. This emphasizes the important role of the hydrophilic domain in amphiphilic proteins when these proteins are incorporated in the cubic phase. Incorporation of the Lipoproteins BamB-E. The change in lattice parameter of the diamond cubic QIID phase upon incorporation of the lipoproteins BamB-E, without their lipid anchor, is shown in Figure 5(C-J). Again, a linear fit through the data points is


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shown as a guide for the eye. A clear linear decrease in QIID lattice parameters with increasing lipoprotein concentration was observed for BamB and BamD with the MAG lipids, Figure 5(C, G). No plateau was observed at the highest concentrations studied: 2 mg mL-1. A slight decrease in QIID lattice parameters was also observed for BamE (Figure 5(I)) with the MAG lipids, no significant trends were observed for BamC (Figure 5(E)). For the branched chain lipids no changes in lattice parameter were observed upon incorporation of any of the BamB-E lipoproteins, as shown by the right column in Figure 5. All data points have been tabulated in Supplementary Information Tables S2.2 – S2.5. For BamB a coexisting primitive cubic QIIP phase was observed at 2 mg mL-1 with MV (lp = 161 Å), MP (lp = 147 Å) and MO (lp 137 Å), as indicated by the circles around the QIID data points in Figure 5(C). The Bonnet ratio of the QIID and QIIP phases observed was ~1.3 for all three lipids, consistent with coexisting phases with similar curvature. However, the primitive cubic QIIP phase is a more hydrated phase with larger water channels than the diamond cubic QIID phase. The transition is expected to reduce geometrical mismatch between the BamB protein and the water channels of the cubic phase, which is expected to be in competition with the increase in curvature as observed by the decrease in lattice parameters of the QIID phase. The effect on QIID lattice parameters with the MAG lipids was most significant for BamB and BamD, with ~3% decrease at 2 mg mL-1 for BamB and ~5% for BamD. BamB has a predicted length of ~45 Å and BamD of ~70 Å based on the crystal structures (Figure 3(B, D)), which is similar in size to, or larger than the cubic phase water channels with 50 – 65 Å diameter for the MAG cubic phases (Table 1). A decrease in QIID lattice parameters (as was observed upon BamB and BamD incorporation) is surprising considering their large hydrophilic domains compared to the water channel diameters. The samples were at 66% w/w hydration which is close to the excess water points of the MAG lipids (47 – 52% w/w hydration). The majority of water within these samples is therefore contained within the water channels


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of the cubic phase, and the mostly hydrophilic BamB-E proteins are expected to be encapsulated within these channels. We note that while the BamB-E proteins are mostly hydrophilic, in vivo they are active at the surface of the cell membrane within the BAM complex. This suggests that there is an effect of lipoprotein incorporation on the polar-apolar interface within the lipidic cubic phase, resulting in an increase in curvature. We suggest that these membrane interactions may be specific to each lipoprotein, as there were significant differences between the effects of the four lipoproteins on the cubic phases. Of the four lipoproteins BamD had the most significant effect on the cubic phase nanostructure. The αhelical BamD (26 kDa), which is the only essential lipoprotein of the BAM complex, may have the strongest interaction with the lipid membrane. Analysis of the primary amino acid sequence for BamA-E using the Protein Data Bank (PDB ID’s are indicated in the caption of Figure 3) and the ExPASy Proteomics service,34 showed that BamD has 8.0% aromatic residues: specifically tryptophan and tyrosine. These amino acids are known to be enriched at the lipid-water interface for proteins embedded in the cell membrane.55,56 Two or more tryptophans co-localized within a peptide were shown to have a significant interaction with the lipid membrane of the MO cubic phase.57 The calculated abundance for BamD is similar to that of aromatic residues found for the transmembrane β-barrel of BamA (7.4%) and the model α-helical transmembrane protein bacteriorhodopsin (bR, PDB ID 4Y9H, 8.4%). The value for BamD is also significantly larger than was found for the soluble PORTA domains of BamA (4.5%), BamB (4.8%), BamC (4.6%), BamE (5.1%) and the model soluble protein Bovine Serum Albumin (BSA, PDB ID 4F5S, 3.7%). The tryptophan and tyrosine amino acid residues in these proteins are illustrated in red in Figure 6. For BamD, as shown in Figure 6(B), a large amount of aromatic side chains can be observed at the surface close to the amino terminal (indicated in magenta) where the lipid-anchor is normally present. The relatively large number of aromatic amino acids at the surface of BamD could potentially explain the strong interaction of this protein with the lipid membrane. BamB had the second most significant effect on the cubic phase nanostructure and is the largest lipoprotein of 17 ACS Paragon Plus Environment

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the family (40 kDa). BamB is well structured as a β-propellor. In contrast, the second largest lipoprotein BamC (35 kDa), is not very well structured (consequently a crystal structure of the full length protein has not been obtained to date). The flexible structure of BamC might explain its reduced impact on the cubic phase nanostructure. BamE is the smallest lipoprotein of the BAM complex (11 kDa, ~20 Å x 30 Å x 12 Å) and potentially does not interact as strongly with the cubic phase because it can easily be accommodated within the water channels of the cubic phases formed by the MAG lipids. Additionally, we note that in vivo BamC and BamE interact with the BAM complex through BamD while BamB and BamD interact with the complex through the PORTA domains of BamA. This could potentially explain why BamB and BamD might interact more strongly with the lipid membrane than BamC and BamE. This interpretation also highlights the importance of a priori knowledge of protein structures and potential interactions prior to studies with cubic phases.


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Figure 6. Secondary structures of model and BAM proteins in green, ordered according to their amount of aromatic tryptophan and tyrosine amino acid residues which are shown in red and the amount is indicated in percent (%). The five amino terminal amino acids are shown in magenta, as this is the attachment site of the lipid-anchor for the lipoproteins. The proteins that are shown are (A) bacteriorhodopsin (bR, PDB ID 4Y9H), (B) BamD (PDB ID 3Q5M), (C) BamA (PDB ID 4K3B), (D) BamE (PDB ID 2KXX), (E) BamB (PDB ID 3Q54), (F) BamC (PDB ID 3TGO), (G) Bovine Serum Albumin (PDB ID 4F5S).

For the branched chain lipids PT and PE no significant changes in nanostructure were observed upon BamB-E protein incorporation. The reduced water channel diameters of 23 – 25 Å for these cubic phases might preclude insertion of the BamB-E proteins (~25 – 70 Å in length) within the aqueous region due to geometrical mismatch. These proteins may locate themselves in the supernatant aqueous phase which surrounds the cubic phase (samples were at 66% w/w hydration, significantly more than the excess water points of 28 – 30% w/w for PT and PE resulting in the cubic phase coexisting with a large excess of water). Alternatively, the increased lateral bilayer pressure in the chain region with the branched chain lipids might make these cubic phase structures less susceptible to changes in curvature. Branched chain lipids are not common in the inner leaflet of the bacterial outer cell membrane, instead this membrane is composed of phospholipids with which BamB-E interact in vivo. Encapsulation of soluble proteins of a similar size to the BamB-E proteins (10 – 40 kDa) within MO has previously been reported to induce a phase transition from the diamond cubic QIID phase to either the more curved primitive cubic QIIP phase or towards the less curved gyroid cubic QIIG phase, depending on the specific protein and conditions.8 Despite significant structural differences between the four lipoproteins BamB-E studied here, they all induced a transition towards a more curved (lower lattice parameter) cubic phase with MAG lipids. This transition may result from specific interactions between these proteins and the polar-apolar interface of the lipid membrane, which we anticipate due to their function in vivo. Lipoproteins are advantageous compared to soluble proteins, in that the lipid anchor can be used to retain a mostly hydrophilic protein within the water channels of the cubic phase under


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the excess water conditions found in vivo. Whilst the lipid anchor was not used in this study, anchored protein molecules can potentially be used in targeted drug delivery and controlled release from cubosome nanoparticles. Previous research on lipoproteins has to date mostly focused on cytochrome c (cyt c) from eukaryotes, which has a highly branched lipid anchor, examining the effect of incorporation within the cubic phase formed by MO or PT. The structural effect of cyt c encapsulation differs significantly between different studies, which can be typical for large bioactives due to sample to sample variability.8,22,58 Using the lipid anchor from the BAM proteins with saturated alkyl chains might be a suitable alternative. The Effect of Cholesterol. The effect of 8% w/w cholesterol on BamA-E incorporation was studied using the MO cubic phase to gain a greater understanding of the effect of membrane rigidity. MO with cholesterol is also commonly used for the in meso crystallization of transmembrane proteins making these conditions relevant to current applications of this material. Data for BamA and BamD with MO with and without 8% w/w cholesterol as an example are shown as an example in Figure 7. BamA and BamD were selected as they were found to have the most significant effect on the MO cubic phase lattice parameters. Data for all BAM proteins with MO + 8% w/w cholesterol is shown in Supplementary Information Tables S2.1 – S2.5. For MO with 8% w/w cholesterol, there was an increase in QIID lattice parameters upon incorporation of BamA with increasing concentrations of BamA (Figure 7(A)) and a decrease in QIID lattice parameters upon incorporation of BamD with increasing concentrations of BamD (Figure 7(B)), as was observed with MO. However, in both cases the effect was slightly reduced in the presence of cholesterol; a 5% increase in lattice parameter instead of 7% with BamA and a 3% decrease in lattice parameter instead of 5% with BamD. We note that, given the noise in the data, this may not be significant. This result is in contrast, however, to what was observed with the α-helical transmembrane butyrate receptors from eukaryotic 20 ACS Paragon Plus Environment

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cells GPR41 and GPR43; with GPR41 and GPR43 there was an increase in lattice parameter with increasing cholesterol concentration in the MO and PT cubic phases.20 For the GPR41 and GPR43 receptors this was explained by an increased uptake of the receptors within cholesterol-doped biomembranes. While eukaryotic cell membranes contain a large amount of cholesterol that rigidifies the lipid bilayer structure, bacterial cell membranes do not typically contain cholesterol.59 For this reason, an increased uptake of the bacterial BamA-E proteins upon the addition of cholesterol was not expected, instead the increased rigidity of the lipid bilayer might explain the reduction in the changes in lattice parameter observed. These results suggest that while for eukaryotic transmembrane proteins adding cholesterol in in meso crystallization trials may be beneficial, this might not be the case for transmembrane proteins from prokaryotes.

D

Figure 7. Incorporation of (A) BamA and (B) BamD into QII of MO (MO, triangles) and MO with 8% w/w cholesterol (MO Chol, squares). The average of top and bottom subwells is plotted (see Supplementary Information Table S2.1, S2.4). A linear fit through the data points is shown as a guide for the eye. BamA was added in 70 mM Tris, 200 mM NaCl and DDM and BamD was added in 70 mM Tris and 200 mM NaCl.


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Comparison of the Effect of BamA-E on the Cubic Phases Formed by Various Lipids. A comparison of the effect of the five BamA-E proteins on the cubic phases formed by MV, MO and PE as representative examples of the six lipid systems studied is shown in Figure 8. As was discussed above, the 0 mg mL-1 values for BamA are 1 – 4 Å higher than for BamB-E because of the presence of the detergent DDM. A straight line at the lattice parameters of QIID with buffer is shown as a reference for BamB-E to allow comparison.

D

Figure 8. Incorporation of BamA-E into QII of (A) MV, (B) MO, (C) PE. The average of top and bottom subwells is plotted (see Supplementary Information Table S2.1-5). BamA was added in 70 mM Tris, 200 mM NaCl and DDM and BamD was added in 70 mM Tris and 200 mM NaCl. with all Bam’s. A straight line is shown at the lattice parameter of the cubic phase with buffer as a reference for BamB-E incorporation.


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For MV (Figure 8(A)), MO (Figure 8(B)) and PE (Figure 8(C)) a similar increase in lattice parameter was found upon BamA incorporation. However, there were differences between the effects of the soluble parts of the lipoproteins BamB-E. For PE, no change in lattice parameter was observed upon BamB-E incorporation. For MO a significant decrease in lattice parameters was observed upon incorporation of the essential BamD protein, but this effect was reduced for BamB, BamC and BamE. For MV a decrease in lattice parameter was observed upon incorporation of all of the lipoproteins. MV has the largest water channel diameter of all cubic phases studied (~65 Å, Table 1), so this might enhance the uptake of the larger BamB and BamC proteins within the cubic phase water channels compared to MO and PE. An increased uptake could lead to an increased interaction of the proteins with the polar-apolar interface of the lipid membrane. Additionally, the excess water point of MV (52% w/w) is significantly higher than for MO (47% w/w) and PE (30% w/w). This means that in case of MV there was only a small excess water region at 66% w/w hydration, which might not be able to retain the BamB-E proteins. The SAXS data show that there were differences upon BamA-E incorporation based on lipid chain length and branching, as well as water channel diameter. For all lipids an increase of ~6 – 8% in lattice parameter was observed upon BamA incorporation. For the lipoproteins BamB-E, no effect on the cubic phases formed by the branched chain lipids was observed, this can be due to the small water channels which might not allow protein incorporation. For the MAG lipids a decrease in lattice parameter upon BamB-E incorporation could be observed, but there were variations based on the specific lipoprotein, as well as the cubic phase water channel diameters. An illustration of BamA, BamB and BamD incorporation into the lipid bilayers of the cubic phase, is shown in the table of contents graphic. This figure also includes the proposed position of the lipid anchor, which was not included in this study.


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CONCLUSIONS In this study, we have analysed the effect of the five proteins from the BAM complex on the nanostructure of the bicontinuous cubic mesophases formed by five different lipids. The lipid systems were three different monoacylglycerols: monovaccenin, monopalmitolein and monoolein, and two branched chain lipids: phytantriol and phytanoyl monoethanolamide. It was found that the amphiphilic transmembrane β-barrel protein BamA caused an increase in the lattice parameter of the cubic phases formed by all five lipids. This effect is expected to be related to the size of the hydrophobic domain relative to the lipid bilayer thickness, as well as the size of the hydrophilic PORTA domains relative to the water channel diameters of the cubic phase. BamB-E are the four lipoproteins of the BAM complex. BamB is the largest lipoprotein of the family and it caused a diamond to primitive cubic phase transition upon encapsulation for the monoacylglycerols. This is expected to reduce geometrical mismatch between the large β-propellor of BamB and the diameter of the water channels, since the primitive cubic phase is a more swollen phase. BamC is the second largest lipoprotein, however, it did not have much effect on the cubic phase nanostructures of the monoacylglycerols. This was explained by its flexible structure and the fact that it interacts through BamD with the BAM complex. The lipo-protein BamD was found to cause a significant decrease in the lattice parameters of the cubic phases formed by monoolein, monopalmitolein and monovaccenin, even though the lipid anchor was removed and the protein has a relatively large diameter relative to the water channels. BamD is the only essential lipoprotein of the four lipo-proteins within the BAM complex. Analysis of the primary sequence of these proteins suggests that BamD can potentially interact with the lipid membrane due to the large amount of aromatic amino acids in the sequence, which are known to be preferentially located at the lipid-water interface. BamE is the smallest lipoprotein of the protein family and did not affect the cubic phase nanostructures significantly. Due to its small size the protein could easily be accommodated within the water channels of the cubic phases formed by the monoacylglycerol lipids. No significant effect on the 24 ACS Paragon Plus Environment

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cubic phase nanostructure was observed for the branched chain lipids upon BamA-E incorporation. The water channels are expected to be too small for encapsulation and there was a significant amount of excess water present in these samples, in which the proteins can reside. An enhanced uptake of lipoproteins was suggested for cubic phases with large water channels, such as for monovaccenin. The lateral bilayer pressure and rigidity of the lipid bilayer, as was shown by the addition of cholesterol, were also found to have an effect. Lipoproteins from the BAM complex could interact with the polar-apolar interface of the lipid membrane, with differences in behavior likely a result of differences in secondary structure and primary amino acid sequence. This is the first time that the effect of a family of proteins within the cubic phase has been systematically studied and that specific interactions of a series of lipoproteins with the lipid membrane were observed for prokaryotic peripheral membrane proteins. These findings indicate that aromatic amino acid residues, and potentially lipid anchors, could facilitate the encapsulation of mostly soluble proteins within the bicontinuous cubic phase; this finding may be of commercial interest for drug delivery and biosensing applications. ACKNOWLEDGEMENTS We thank Drs. Adrian M. Hawley, Stephen T. Mudie and Nigel M. Kirby for their 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 the robotic setup of plates at the C3 Collaborative Crystallization Centre, CSIRO, Parkville, Australia. A/Prof. Sally L. Gras is supported by the ARC Dairy Innovation Hub IH120100005. Dr. Charlotte Conn is the recipient of an ARC DECRA Fellowship DE160101281. Dr. HsinHui Shen is supported by the NMHRC CDF GNT1106798. Leonie van ‘t Hag is supported by a Melbourne International Research Scholarship and a CSIRO PhD scholarship. Prof. Ian Henderson provided the BamB-E plasmids. The authors acknowledge and respect Professor Toyoki Kunitaki for his immense


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contributions to the field of molecular self-assembly. He has served as a role model and as an inspiration to many researchers across the globe. SUPPLEMENTARY INFORMATION (1) Figures showing reproducibility SAXS results. (2) Tables showing all phases and lattice parameters (lp) for MV, MP, MO (+8% w/w cholesterol), PT and PE with BamA-E at all protein concentrations studied. (3) Method to calculate the bilayer thickness and water channel diameter. REFERENCES (1) Buchanan, S. K.; Noinaj, N. The BAM Complex Methods and Protocols. Bam Complex: Methods and Protocols 2015, 1329, V-Vi. (2) Landau, E. M.; Rosenbusch, J. P. Lipidic cubic phases: A novel concept for the crystallization of membrane proteins. Proceedings of the National Academy of Sciences of the United States of America 1996, 93, 14532-14535. (3) Seddon, A. M.; Curnow, P.; Booth, P. J. Membrane proteins, lipids and detergents: not just a soap opera. Bba-Biomembranes 2004, 1666, 105-117. (4) Almsherqi, Z. A.; Landh, T.; Kohlwein, S. D.; Deng, Y. R. Cubic Membranes: The Missing Dimension of Cell Membrane Organization. Int Rev Cel Mol Bio 2009, 274, 275-342. (5) Luzzati, V.; Tardieu, A.; Gulikkrz.T; Rivas, E.; Reisshus.F. Structure of Cubic Phases of Lipid-Water Systems. Nature 1968, 220, 485-488. (6) Zabara, A.; Negrini, R.; Onaca-Fischer, O.; Mezzenga, R. Perforated Bicontinuous Cubic Phases with pH-Responsive Topological Channel Interconnectivity. Small 2013, 9, 3602-3609. (7) Zabara, A.; Negrini, R.; Baumann, P.; Onaca-Fischer, O.; Mezzenga, R. Reconstitution of OmpF membrane protein on bended lipid bilayers: perforated hexagonal mesophases. Chem Commun 2014, 50, 2642-2645. (8) Conn, C. E.; Drummond, C. J. Nanostructured bicontinuous cubic lipid self-assembly materials as matrices for protein encapsulation. Soft Matter 2013, 9, 3449-3464. (9) Angelova, A.; Angelov, B.; Mutafchieva, R.; Lesieur, S.; Couvreur, P. Self-Assembled Multicompartment Liquid Crystalline Lipid Carriers for Protein, Peptide, and Nucleic Acid Drug Delivery. Accounts Chem Res 2011, 44, 147-156. (10) Caffrey, M. A comprehensive review of the lipid cubic phase or in meso method for crystallizing membrane and soluble proteins and complexes. Acta Crystallographica Section F-Structural Biology Communications 2015, 71, 3-18. (11) Vallooran, J. J.; Handschin, S.; Pillai, S. M.; Vetter, B. N.; Rusch, S.; Beck, H.-P.; Mezzenga, R. Lipidic Cubic Phases as a Versatile Platform for the Rapid Detection of Biomarkers, Viruses, Bacteria, and Parasites. Advanced Functional Materials 2016, 26, 181-190. (12) Nazaruk, E.; Smolinski, S.; Swatko-Ossor, M.; Ginalska, G.; Fiedurek, J.; Rogalski, J.; Bilewicz, R. Enzymatic biofuel cell based on electrodes modified with lipid liquid-crystalline cubic phases. J Power Sources 2008, 183, 533-538. (13) Mulet, X.; Boyd, B. J.; Drummond, C. J. Advances in drug delivery and medical imaging using colloidal lyotropic liquid crystalline dispersions. J Colloid Interf Sci 2013, 393, 1-20. 26 ACS Paragon Plus Environment

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(14) van 't Hag, L.; Knoblich, K.; Seabrook, S. A.; Kirby, N. M.; Mudie, S. T.; Lau, D.; Li, X.; Gras, S. L.; Mulet, X.; Call, M. E.; Call, M. J.; Drummond, C. J.; Conn, C. E. Exploring the in meso crystallization mechanism by characterizing the lipid mesophase microenvironment during the growth of single transmembrane α-helical peptide crystals. Philosophical Transactions of the Royal Society of London A: Mathematical, Physical and Engineering Sciences 2016, 374. (15) Phan, S.; Fong, W. K.; Kirby, N.; Hanley, T.; Boyd, B. J. Evaluating the link between selfassembled mesophase structure and drug release. Int J Pharmaceut 2011, 421, 176-182. (16) Zabara, A.; Mezzenga, R. Controlling molecular transport and sustained drug release in lipid-based liquid crystalline mesophases. J Control Release 2014, 188, 31-43. (17) Angelov, B.; Angeova, A.; Papahadjopoulos-Sternberg, B.; Hoffmann, S. V.; Nicolas, V.; Lesieur, S. Protein-Containing PEGylated Cubosomic Particles: Freeze-Fracture Electron Microscopy and Synchrotron Radiation Circular Dichroism Study. J Phys Chem B 2012, 116, 7676-7686. (18) Conn, C. E.; Darmanin, C.; Sagnella, S. M.; Mulet, X.; Greaves, T. L.; Varghese, J. N.; Drummond, C. J. Incorporation of the dopamine D2L receptor and bacteriorhodopsin within bicontinuous cubic lipid phases. 1. Relevance to in meso crystallization of integral membrane proteins in monoolein systems. Soft Matter 2010, 6, 4828-4837. (19) van't Hag, L.; Shen, H. H.; Lu, J. X.; Hawley, A. M.; Gras, S. L.; Drummond, C. J.; Conn, C. E. Deconvoluting the Effect of the Hydrophobic and Hydrophilic Domains of an Amphiphilic Integral Membrane Protein in Lipid Bicontinuous Cubic Mesophases. Langmuir 2015, 31, 12025-12034. (20) Liang, Y. L.; Conn, C. E.; Drummond, C. J.; Darmanin, C. Uptake of the butyrate receptors, GPR41 and GPR43, in lipidic bicontinuous cubic phases suitable for in meso crystallization. J Colloid Interf Sci 2015, 441, 78-84. (21) van 't Hag, L.; Li, X.; Meikle, T. G.; Hoffmann, S. V.; Jones, N. C.; Pedersen, J. S.; Hawley, A. M.; Gras, S. L.; Conn, C. E.; Drummond, C. J. How Peptide Molecular Structure and Charge Influence the Nanostructure of Lipid Bicontinuous Cubic Mesophases: Model Synthetic WALP Peptides Provide Insights. Langmuir 2016, DOI:10.1021/acs.langmuir.6b01058. (22) Mazzoni, S.; Barbosa, L. R. S.; Funari, S. S.; Itri, R.; Mariani, P. Cytochrome-c Affects the Monoolein Polymorphism: Consequences for Stability and Loading Efficiency of Drug Delivery Systems. Langmuir 2016, 32, 873-881. (23) Noinaj, N.; Rollauer, S. E.; Buchanan, S. K. The beta-barrel membrane protein insertase machinery from Gram-negative bacteria. Curr Opin Struc Biol 2015, 31, 35-42. (24) Kleinschmidt, J. H. Folding of beta-barrel membrane proteins in lipid bilayers Unassisted and assisted folding and insertion. Bba-Biomembranes 2015, 1848, 1927-1943. (25) Noinaj, N.; Kuszak, A. J.; Gumbart, J. C.; Lukacik, P.; Chang, H. S.; Easley, N. C.; Lithgow, T.; Buchanan, S. K. Structural insight into the biogenesis of beta-barrel membrane proteins. Nature 2013, 501, 385-+. (26) Tamm, L. K.; Hong, H.; Liang, B. Y. Folding and assembly of beta-barrel membrane proteins. Bba-Biomembranes 2004, 1666, 250-263. (27) Wimley, W. C. The versatile beta-barrel membrane protein. Curr Opin Struc Biol 2003, 13, 404-411. (28) Dong, C.; Yang, X.; Hou, H. F.; Shen, Y. Q.; Dong, Y. H. Structure of Escherichia coli BamB and its interaction with POTRA domains of BamA. Acta Crystallogr D 2012, 68, 1134-1139. (29) Kim, K. H.; Aulakh, S.; Paetzel, M. Crystal Structure of beta-Barrel Assembly Machinery BamCD Protein Complex. J Biol Chem 2011, 286, 39116-39121. (30) Dong, C.; Hou, H. F.; Yang, X.; Shen, Y. Q.; Dong, Y. H. Structure of Escherichia coli BamD and its functional implications in outer membrane protein assembly. Acta Crystallogr D 2012, 68, 95101.


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Effect of Lipid-Based Nanostructure on Protein Encapsulation within

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