Lipidic Cubic Phase-Induced Membrane Protein Crystallization

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Lipidic Cubic Phase-Induced Membrane Protein Crystallization: Interplay Between Lipid Structure, Mesophase Properties, and Crystallogenesis Alexandru Zabara, Thomas Geoffrey Meikle, Raphael Trenker, Shenggen Yao, Janet Newman, Thomas S. Peat, Frances Separovic, Charlotte E Conn, Melissa J. Call, Matthew E. Call, Ehud M. Landau, and Calum J. Drummond Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.7b00519 • Publication Date (Web): 16 Aug 2017 Downloaded from http://pubs.acs.org on August 21, 2017

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Crystal Growth & Design

Lipidic Cubic Phase-Induced Membrane Protein Crystallization:

Interplay

Between

Lipid

Molecular Structure, Mesophase Structure and Properties, and Crystallogenesis Alexandru Zabara,a,f,† Thomas G. Meikle,b,e,† Raphael Trenker,c,d Shenggen Yao,e Janet Newman,f Thomas S. Peat,f Frances Separovic,b Charlotte E. Conn,a Melissa J. Call,c,d Matthew E. Call,c,d Ehud M. Landau,g Calum J. Drummond.a,* a

School of Science, College of Science, Engineering and Health, RMIT University,

124 La Trobe Street, Melbourne, VIC 3000, Australia. b

School of Chemistry, Bio21 Institute, University of Melbourne, VIC 3010, Australia

c

Structural Biology Division, The Walter and Eliza Hall Institute of Medical Research,

Parkville, VIC 3052, Australia d

Department of Medical Biology, University of Melbourne, VIC 3010, Australia

e

Bio21 Institute, University of Melbourne, VIC 3010, Australia

f

Biomedical Manufacturing Program, Commonwealth Scientific and Industrial Research

Organization (CSIRO), 343 Royal Parade, Parkville, VIC 3052, Australia. g

Department of Chemistry, University of Zurich, 8057 Zurich, Switzerland.

*Correspondence to: Professor Calum J. Drummond

ACS Paragon Plus Environment

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School of Science RMIT University, GPO Box 2476, Melbourne, VIC 3001, Australia Tel: +61 3 9925 4265 E-mail: [email protected] †

These authors have contributed equally to this work.

ABSTRACT: Obtaining well-diffracting crystals of membrane proteins, which is crucial to the molecularlevel understanding of their intrinsic three-dimensional structure, dynamics and function, represents a fundamental bottleneck in the field of structural biology. One of the major advances in the field of membrane protein structural determination was the realization that the nanostructured lipidic cubic phase (LCP) environment constitutes a membrane mimetic matrix that promotes solubilization, stabilization, and crystallization of specific membrane proteins. Despite two decades passing since the introduction of LCP-based membrane protein crystallization, the research community’s understanding of the processes that drive protein nucleation and macromolecular assembly in the LCP generally remains limited. In the current study, we present a detailed, systematic investigation into the relationship between the chemical structure of the lipid, the physical properties of the ensuing mesophase, the translational diffusion of the encapsulated membrane protein, and resulting protein crystallization. Importantly, we show for the first time that cubic phase transport properties directly modulate the size and morphology of fully-grown protein crystals without affecting their crystallographic space group. These findings provide a deeper understanding of the LCP-based crystal growth process, setting the stage for the engineering of membrane protein crystals at the molecular level via intentional design of the host cubic phase, and, ultimately, accelerating progress in the field of membrane protein structural biology.


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INTRODUCTION: Research into the structure, function and dynamics of membrane proteins and their complexes has skyrocketed in the past few decades due to their inherent biomedical significance, great diversity and ubiquitous occurrence, and their importance as drug targets. They represent the target for more than 40% of all commercially available drugs.1 This class of biomolecules, comprising roughly 20-30% of the total protein population in most organisms,2 has proved significantly more difficult to address experimentally than the complementary class of soluble proteins, presenting various challenges and hurdles in the quest to determine their structure, dynamics and intricate molecular mechanisms. To date, close to 650 unique three dimensional structures of these biomolecules have been deposited in the protein data bank3 as compared with approximately 100,000 structures of soluble proteins. The unique function of membrane proteins, as well as their interactions with various other cellular players in health and disease, are directly governed by, and can be elucidated from, their three-dimensional structures, which are adopted during biosynthesis and insertion into the cellular membrane.

A detailed atomic understanding of a protein’s structure can,

therefore, offer the possibility of deliberately altering its functionality to either implicitly negate or promote its molecular activity. X-ray crystallography has been the method of choice in obtaining such high-resolution structures but this method requires the availability of well-ordered three dimensional crystals, which has been the major bottleneck slowing progress in the field. The initial approach towards overcoming this problem employed the use of detergents, aimed at shielding the hydrophobic trans-membrane domain of the protein molecules during vapor diffusion-based crystallization experiments, and has yielded the first crystals and high resolution structures of membrane proteins.4, 5 Two decades ago the fields of lyotropic liquid crystal (LLC) mesophase chemistry and membrane protein structural


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biology converged, leading to the utilization of the structural and dynamic properties of lipidic cubic phases (LCPs) to provide a membrane-mimetic host matrix for the stabilization, solubilization and crystallization of these inherently unstable biomacromolecules.6 This novel approach exploits the innate ability of the LLC bilayer to provide optimal shielding for the protein hydrophobic domain, as well providing viscoelastic and structural properties similar to the cellular membrane,7,

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and hence a closer biomimetic environment than detergent

micelles for membrane protein reconstitution. The LCP is a highly curved, three dimensional periodic liquid crystalline structure that spontaneously self assembles across a range of lipid hydrations, and has shown great potential for applications in a number of fields ranging from drug delivery and food science to structural biology.9-14 To this point, the relationship between the composition and structure of the cubic phase and the diffusion of soluble bioactives has been well established,15-17 while the cubic phase bilayer has been shown to assist retention of functionality for large integral membrane proteins.18,

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Moreover, as was demonstrated for hydrophilic proteins, by

overcoming the intrinsically small aqueous channel size of the cubic phase and thereby altering the phase transport properties, one can potentially allow for a much wider array of proteins to be crystallized as well as potentially engineer crystal growth at the molecular level.20, 21 Since its initial utilization, the field of LCP-based crystallization of membrane proteins has thrived,22-24 transforming the field and enabling the determination of ~260 high-resolution structures3 for a number of previously unknown membrane proteins,25,

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including the

pharmacologically relevant class of G-Protein Coupled Receptors.27-29 A related, alternative in-meso crystallization approach employed the use of a disordered sponge phase for successful membrane protein crystal growth.30-32 The sponge phase retains the fundamental bilayer lipid membrane structure but with the loss of the periodic order in three dimensions


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associated with the LCP. Moreover, crystallization of small hydrophilic proteins from the aqueous channels of two distinct LLC symmetries, the double diamond cubic phase and the inverse hexagonal phase, revealed the possibility of employing the various existing symmetries in order to engineer crystal growth of soluble species at the molecular level.33-35 Despite significant progress in developing high-throughput methodologies for sample preparation, LLC structural analysis and crystal growth,36, 37 our fundamental understanding of the direct interplay between the structural and molecular properties of the lipid mesophase and the process of membrane protein crystal growth remains poorly developed and calls for further investigation. In previous work, we have examined the role of lipid nanosctucture and phase in the crystallization of membrane proteins, using synchrotron SAXS to probe phase changes occurring over the first hours of crystallization in meso.38 Multiple studies now point to the mechanistic importance of a localized lamellar phase, which forms at the site of crystal nucleation,38, 39 but the properties and conditions contributing to protein crystal quality, size and speed of growth remain poorly defined. In this study we aim to elucidate the interplay between the lipid chemical and nanoscale structure, the ensuing cubic phase transport properties (both within the hydrophilic aqueous channels and the hydrophobic lipid bilayer) and the process of membrane protein crystal nucleation and growth, examining factors such as crystal size, speed of formation, and

morphology. To achieve this, we have investigated a range of monoacylglycerols (MAGs) with various chain lengths and position of the cis double bond, as well as an isoprenoid-type branched chain lipid, phytantriol (PT), as building blocks of the host matrices for crystallization of membrane proteins. The membrane protein used in this study was the E. coli virulence factor intimin, selected for its ease of expression and purification, and previous successful crystallization from LCPs. Physicochemical characterization techniques, including pulsed-field gradient NMR, fluorescence recovery after photobleaching (FRAP) and small


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angle X-ray scattering (SAXS) with microscopy-based visual inspections, were combined with single crystal synchrotron X-ray diffraction in order to achieve a comprehensive understanding of how the crystallization process is affected by the molecular and bulk properties of the host phase. The results provide a significant advancement in our fundamental understanding of LCP-based crystallization and, by extension, assist the ongoing quest to increase the number of high-resolution membrane protein structures. MATERIALS AND METHODS: Materials: Monoolein (MO), monopalmitolein (MP), monovaccenin (MV) and monoeicosenoin (ME) were

purchased

from

Nu-Chek

Prep

(MN,

USA).

3,7,11,15-tetramethyl-1,2,3-

hexadecanetriol (phytantriol) (96%) was provided by DSM Nutritional Products, Germany. LCP glass plates with a 100 µm double sided tape spacer and 200 µm plastic seals were purchased from Molecular Dimensions. Salts used for the preparation of crystallization buffers were purchased from Sigma Aldrich (Sydney, Australia), unless otherwise stated. Expression and purification of intimin: Information detailing the construction of the plasmids containing the intimin E. coli O157:H7 gene has been provided previously.40 The construct was kindly provided by Dr Susan K. Buchanan from the National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, USA, and was used with no further modifications. An in depth description of the expression and purification of the membrane protein intimin is provided in the Supporting Information. Briefly, the vector containing the E. coli O157:H7 gene was transformed into BL21 (DE3) cells which were grown in Terrific Broth media (50 µg/mL kanamycin) and lysed using a probe sonicator (Misonix S4000), using 30 s bursts at 60% amplitude. Membranes containing the intimin protein were harvested using ultra-


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centrifugation (160,000 g, 60 min, 4°C) and resuspended in solubilization buffer (50 mM TRIS pH 8.0, 200mM NaCl, 20 mM imidazole, 5% Elugent (Calbiochem)) by stirring overnight at 4°C. Insoluble material was subsequently removed from the sample by ultracentrifugation (250,000 g, 120 min, 4°C). Protein purification was conducted using a combination of affinity and ion exchange chromatography, with protein containing fractions concentrated using a YM30 Amicon Ultra concentrator (Millipore). Sample preparation for benchtop SAXS experiments: SAXS samples were prepared by gently melting the lipids and mixing with a water/protein sample using a coupled syringe apparatus, resulting in formation of the mesophase. The water content used in SAXS samples was selected to approximate the excess water point for each lipid, ensuring formation of the QIID phase commonly used in crystallization experiments. Water contents were as follows: MO + 42 w/w% H2O, MP + 50 w/w% H2O, MV + 50 w/w% H2O, ME + 40 w/w% H2O, 7.7 MAG + 50 w/w% H2O, PT + 30 w/w% H2O. A needle was attached to the syringe and the mesophase injected into a 1.5 mm glass capillary, which was then sealed using wax. Samples were equilibrated overnight prior to performing measurements. SAXS diffraction patterns were recorded at 20°C on a Bruker MicroCalix SAXS/WAXS system and analysed using the IDL based AXCESS software package, developed at Imperial College London.41 Pulsed-field gradient (PFG) NMR: Lipid mesophase samples used in PFG-NMR experiments were prepared in the same fashion as SAXS samples. The water content used in each NMR experiment was as follows: MO + 40 w/w% H2O, MP + 50 w/w% H2O, MV + 48 w/w% H2O, ME + 46 w/w% H2O, PT + 25 w/w% H2O. The mesophase was injected into a 2 mm OD glass capillary and allowed to


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equilibrate overnight before being placed into a 5 mm NMR tube filled with either D2O or CDCl3 and fixed in place using a Teflon adapter. The translational diffusion coefficients of lipids were measured on a Bruker Avance II 800 MHz spectrometer equipped with a TCI cryoprobe using a standard BPP-STE sequence without modification,42, 43 as described previously.17 Samples were measured at 20°C. The diffusion coefficient, D, was determined by fitting diffusion weighted intensities of well resolved peaks to the following equation using the T1/T2 relaxation module in TOPSPIN as well as OriginPro software:

δ

τ

= exp {−γ δ − − } 3 2

(1)

where γ is the gyromagnetic ratio of protons and s, g, δ, and ∆ are the shape factor, amplitude, duration and separation, respectively, of the single pair of gradient pulses, while τ is the time interval within the bipolar pulse pair. Sinusoidal shaped gradient pulses, with of 2.5 ms (except for the phytantriol sample where = 4 ms), ∆ of 50 ms and the amplitude of the field Gz ranging from 2.8 to 53.1 (G cm-1) were used. To calibrate the strength of pulsed-field gradient, Gz, the self-diffusion coefficient of residual H2O in a 100% 2H2O sample was measured at 298.13 K.44, 45 Fluorescence recovery after photo-bleaching (FRAP): Purified intimin was labeled with Sulfo-Cyanine3 NHS ester (lumiprobe) in 0.1 M sodium phosphate pH 8.4 according to the manufacturer’s protocol with a protein concentration of 2 mg/ml. Free dye was removed using a Micro Bio-spinTM 6 column (Bio-Rad) following the buffer exchange protocol (50 mM Tris pH 8, 0.1% n-dodecyl-β-D-maltopyranoside). The cubic phases were prepared as described above using a final labelled protein concentration of 1 mg/ml. The samples were prepared on microscopy slides with 140 µm thick double-sided


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adhesive spacers, commonly used as LCP crystallization sandwich plates, and sealed with glass coverslips. The buffer mentioned above was used as precipitant solution for each measurement. FRAP experiments were performed on a Zeiss LSM 780 confocal microscope at 514 nm excitation bleaching a small region of interest (ROI) with a radius of 27 µm. Five pre-bleach images were recorded, the ROI was bleached and recovery was recorded for a total of 6-7 min in 0.78 s intervals. The fractional fluorescence recovery was obtained by normalizing the mean fluorescence signal in the ROI to pre-bleach intensities and potential bleaching effects during the measurement were compensated by subtracting background fluorescence changes in non-bleached areas as previously described.46-48 High-throughput crystallization of intimin: High-throughput LCP preparation and crystallization was carried out according to literature.14 The protein solution was mixed with molten lipid to achieve a water content of 50% (v/v) for MP, 42% (v/v) for MO, 50% (v/v) for MV, 40% (v/v) for ME, 70% (v/v) for 7.7 MAG and 30% (v/v) for PT. The LCP was dispensed onto the surface of a LCP glass plate with doublesided tape spacer (Laminex 100 µm spacer, Molecular Dimensions) in 200 nL aliquots, 1 µL of screen was dispensed on top, and a 100 µm plastic seal (Molecular Dimensions) was applied to seal the experiment. The screen was designed around three previously known conditions for intimin crystallization (Table S1), randomized to ensure reproducibility, and with the most successful conditions selected for presentation. Both the LCP and screen were dispensed using a Mosquito LCP machine equipped with a humidity chamber (TTPLabtech, UK). Sealed plates were incubated at 20°C and imaged in a Minstrel HT/UV imaging system (Rigaku). The resulting crystals were harvested into Mylar loops and cryo-cooled using liquid nitrogen. Data were collected at the MX2 microfocus beamline at the Australian Synchrotron using one


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degree oscillations for a total of 360 degrees of data. The data were indexed with XDS49 and scaled using Aimless.50 For the one data set with reflections beyond 3 Å (MP lipid), molecular replacement was performed with Phaser,51 using the PDB entry 4e1s as the search model. Manual building was done using Coot52 and refinement was performed using both Refmac53 and Phenix Refine54 with the dictionary cif file for MP generated by eLBOW. The PDB deposition code for this structure is 5g26. RESULTS: Small angle X-ray scattering investigation of the cubic phase nanostructure under crystallization conditions Mismatch between the length of the lipid bilayer and the membrane protein hydrophobic domain can potentially induce irreversible structural changes within the mesophase symmetry. Therefore, in order to correlate the specific diffusive properties of the various phases with protein crystal nucleation and growth, retention of the double diamond LCP structural symmetry subsequent to protein reconstitution needs to be initially confirmed for each lipidic system. To this end, we chose specific formulations for each system whereby the respective binary lipid:water phase diagrams (i.e., devoid of added membrane protein) would yield a LCP with QIID architecture. The structure of the ensuing mesophase, with and without reconstituted intimin, was investigated by SAXS. In all cases investigated, peaks were observed in the specific spacing ratio √2, √3, √4, √6, √8, √9, representative of the QIID diamond cubic phase (crystallographic space group Pn3m – see S.I. Figure S1- S5). Structural parameters, including the bilayer thickness and water channel diameter, determined from SAXS (see S.I. for method) for the various LCPs with and without addition of intimin protein in solution at a concentration of 20 mg/mL are shown in Table 1.


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Regardless of the choice of lipid, addition of 20 mg/mL intimin solution did not have a significant effect on the lattice parameter, water channel diameter and bilayer length of the host cubic phase, despite the bilayer thickness varying between 27 Å (phytantriol) to 37 Å (ME).

The retention of the cubic phase nanostructure following protein reconstitution,

coupled with the lack of variability of the structural parameters, suggests that hydrophobic mismatch between the protein hydrophobic domain and the lipid bilayer thickness in these systems has less of an effect than observed previously for other peptides and proteins.18, 55 Thus the reconstitution of intimin did not destabilize the structure and symmetry of the host mesophase, despite the different degrees of hydrophobic mismatch between the bilayer thickness and the protein hydrophobic core. Cubic symmetry, with or without addition of protein, was not observed in samples consisting of intimin reconstituted within the bilayer of 7.7 MAG, the MAG lipid with the shortest aliphatic tail and bilayer thickness. The system exhibited a lamellar or disordered sponge phase (depending on temperature and hydration) which cannot be directly compared with the QIID phase adopted by the other lipid systems (Figure S6).

Sample ME 40% H2 O MV 50% H2 O MO 42% H2 O MP 50% H2 O PT 30% H2 O

Lattice Parameter (Å)

Water Water Channel Diameter (Å)

Protein (20 mg/mL) Lattice Water Bilayer Parameter Channel Thickness (Å) Diameter (Å) (Å)

Bilayer Thickness (Å)

112.5

49.9

38.1

109.0

48.3

37.0

127.2

64.2

35.3

126.7

63.9

35.1

106.4

48.4

34.8

104.8

47.7

34.2

118.3

60.4

32.1

118.6

60.6

32.2

68.1

25.6

27.7

66.6

25.0

27.1

Table 1: The effect of protein reconstitution on the symmetry and structural parameters of various host LCPs. All mesophase materials shown exhibited a QIID cubic


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phase (crystallographic space group Pn3m), and are listed in order of decreasing bilayer thickness.

Exploring lipid self-diffusion via pulsed-field gradient NMR Pulsed-field gradient NMR was used to determine the lipid translational diffusion coefficients of MP, MO, MV, ME and PT when each was reconstituted with pure water to form the QIID cubic phase. Sample inhomogeneity precluded PFG-NMR measurements in the case of 7.7 MAG. The apparent diffusion coefficients range from 2.0 x 10-12 m2s-1 (PT) to 1.8 x 10-11 m2s-1 (MP), and are presented in Figure 1 as a function of bilayer thickness, which was calculated from SAXS data for pure lipid QIID excess water mesophase systems. Values of MO and PT lipid diffusion closely match those observed for similar LCP samples in a previous study.17 The diffusion of PT was found to be significantly slower than that observed for MAG lipids, while diffusion coefficients for the MAGs decreased in the order MP > MO ≈ MV > ME. Amongst MAG lipids, we note an inverse correlation between the bilayer thickness of the lipid mesophase and the translational diffusion coefficient of the lipid; with the shortest chain lipids diffusing fastest. To our knowledge, this is the first time that this effect has been reported for these lipid systems.


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Figure 1. Plot of lipid diffusion coefficients determined experimentally using PFG-NMR versus bilayer thickness, calculated from SAXS measurements of pure lipid excess water mesophase systems. Monitoring diffusion of membrane protein molecules in the LCP via FRAP Cy3-labelled intimin was reconstituted into the LCPs of five different host lipids under otherwise identical conditions, and the diffusion characteristics of the protein in the various LCP media compared by FRAP. All recovery curves (Figure S7) could be described using a one-component diffusion model with mean diffusible fractions between 72 and 88%. The fastest recovery rate was observed in MP with a mean diffusion coefficient of 7.5×10-12 m2/s, which is higher than the diffusion coefficients of 7×10-12 and 6.9×10-12 m2/s obtained in MO and MV, respectively (Figure 2). Protein diffusion in ME and PT were significantly slower with mean values for the diffusion coefficient of 5.2×10-12 and 2×10-12 m2/s, respectively.


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Figure 2. Plot of experimentally determined diffusion coefficients of intimin reconstituted in a series of MAG lipids and phytantriol versus mesophase bilayer thickness, calculated from SAXS measurements of pure lipid excess water mesophase systems. Error bars indicate ± 1 SD. We note a strong correlation between the diffusion coefficient of lipid molecules and the protein diffusion coefficient, which decreases in the order MP > MO ≈ MV > ME > PT. This is not unexpected as intimin, a hydrophobic protein, should reside mainly within the bilayer. As with lipid molecule diffusion, the diffusion of intimin reconstituted in the various MAG bilayers could be directly linked with mesophase bilayer thicknesses; membrane protein molecules are found to diffuse faster within the shorter chained lipids such as MP, compared to lipids with longer aliphatic tails such as MV or ME. Diffusion within the bilayer composed of PT, which features a branched saturated hydrocarbon tail, was significantly slower than in MAG bilayers. This may result from increased stearic interactions and lateral pressure within the bilayer, indicating the inefficacy of PT as a membrane protein hosting matrix throughout the process of LCP-based crystallization. Monitoring the effects of host LCP properties on intimin crystallization


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Crystallization trials were designed to assess the effect of lipid and protein diffusion on protein crystal growth and morphology. Crystal growth was also observed from the 7.7 MAG matrix, although we note that the results cannot be directly compared with the growth from the other lipid matrices due to the induced transition from a cubic to a disordered sponge phase.

Figure 3. Overview of LCP-based crystallization setups of intimin using the following LCPs: 7.7 MAG, MP, MO, MV, PT and ME. Images were obtained using an automated Minstrel HT/UV imaging system (Rigaku). In the case of ME, the observed aggregation and crystal formation in the drop is a result of lipid crystallization, with none of the formed crystals showing UV absorbance. Representative images of intimin crystals, grown from the lipidic cubic mesophase systems, are presented in Figure 3. Image data sets for MO, MP, MV and ME based experiments were collected in a complementary study, which offers a preliminary analysis of lipid effects on crystallization.38 We note that the crystal morphology in each system was highly reproducible


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and strongly dependent on the lipid used. To increase the likelihood of obtaining protein

crystals from of each lipid mesophase, a randomized, 96 condition screen was used to induce crystallization (See Table S1 for full details). Crystallization occurred across a range of screen conditions. Optimal screen conditions, resulting in the largest crystals (those presented in Fig. 3), were similar for each lipid, and are as follows: 79 mM MgCl2, 33.7% v/v PEG 400, 100 mM NaCl, 100 mM trisodium citrate-citric acid, pH 4.59 for MO crystallization; 143 mM MgCl2, 25.4% v/v PEG 400, 65 mM NaCl, 100 mM trisodium citrate-citric acid, pH 4.96 for MP crystallization; 142 mM MgCl2, 34.7% v/v PEG 400, 92 mM NaCl, 100 mM trisodium citrate-citric acid, pH 5.6 for MV crystallization; and 84 mM MgCl2, 26.2% v/v PEG 400, 99 mM NaCl, 100 mM trisodium citrate-citric acid, pH 4.92 for 7.7 MAG crystallization.

In each lipid mesophase the screen conditions leading to optimal crystallization contained identical compounds present in similar concentrations. We assume, based upon previous studies,56,

57

and the large difference in crystal morphology between lipids, that small

variations in screen component concentrations exert only minor influence on the overall thermodynamics and kinetics of intimin protein crystallization, and thus allow for adequate comparison between different lipids. Specifically, thin, needle-shaped crystal clusters were consistently observed in both MP- and MO-based crystallization trials. The crystals growing from within the MP cubic phase were much longer and the time needed for nucleation and maximum crystal growth was considerably shorter (Table 2). These were first observed after less than 24 hours and reached their maximum size after approximately 8 days. In the case of MO, however, the first crystals were observed after approximately 48 hours and reached maximum size after 11 days from the onset of crystallization.


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Changing the position of the cis double bond within the aliphatic tail (MO vs. MV) and as a result changing the physico-chemical properties of the lipidic system resulted in two distinct crystal growth behaviours. In the case of MO, relatively fast growing, needle-shaped crystals were consistently observed, while star-like clusters of thin crystal plates were found throughout the MV-based crystallization trials. Both the MV based star shaped crystals, and the needle shaped crystals grown from MO required a similar amount of time to reach their maximum size, at 12 and 11 days respectively. In spite of exhaustive efforts, no crystals were observed from either ME- or PT-based LCPs. We suggest that the larger hydrophobic mismatch between the protein molecules and the lipid bilayer, coupled with the significantly slower diffusion of both protein and lipid molecules, impeded nucleation and prevented crystal growth. Moreover, previous observations revealed that PT is a poor host for membrane protein and peptide reconstitution, consistent with our observations.55 Interestingly, reconstitution and crystallization of intimin from within the 7.7 MAG bilayer resulted in thick, rectangular shaped crystals that first appeared after less than 24 hours and required approximately 3 days to reach maximum growth. In spite of losing its structural integrity, the 7.7 MAG mesophase yielded the thickest crystals in the shortest amount of time, when compared with the other tested lipids. Diffusion of proteins and solutes within this phase is significantly faster than all the other tested lipids (see S.I. Figure S7), which may enable the observed behaviour. Given the results of these crystallization experiments, we conclude that the rate of crystal growth is directly related to lipid bilayer thickness and, therefore, with lipid and solute diffusion coefficient rates for the MAG lipids. Crystal nucleation is generally faster, and the maximum crystal size is reached earlier, for the shortest chain lipids. Moreover, the physiologically less fitting, “adverse” environment provided by the PT branched chain lipid


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bilayer, coupled with diffusive properties that approach one order of magnitude slower than for other lipidic systems, contribute to the inefficiency of the PT-based mesophase as a host matrix for membrane protein reconstitution and crystallization. The obtained LCP-based crystals proved to be of good diffraction quality, with the best being the MP-grown crystals diffracting to a resolution of 2.42 Å, and in all cases confirming the expected beta-barrel structure of the membrane protein. While the morphology of the fully grown crystal varied widely between the various systems, the space group (C2221) was identical in all cases, consistent with that previously determined for intimin (Table 3).40 Unsurprisingly, the structure determined in the presence of MAG lipids is highly similar to the structure previously determined by the Buchanan lab using a MO-based cubic phase (PDB code 4E1S).40 The dimensions of the unit cells were approximately 116 Å × 120 Å × 39 Å for all lipids, with the exception of 7.7 MAG and MV where the X dimension was slightly lower (108 Å). Despite their different morphologies, the intimin crystals obtained from the different cubic phases allow for a very similar packing of the protein molecules (Figure S8). This suggests that the primary driving force behind LCP-based crystal formation and growth is the diffusion rate of protein and solute molecules within the 3D matrix of the LCP, which may relate, in the case of hydrophobic proteins, to the degree of mismatch between the bilayer thickness and the width of the transmembrane domain of the protein. The choice of lipid hosting media, and thus the specific transport rates of the formed cubic phases, therefore, drive the kinetics, morphology and growth rate of the protein crystal without altering the internal packing of the molecules or the space group of the formed crystal. Table 2. Overview of the time required by the intimin molecules within each of the six tested lipidic phases from protein reconstitution to microcrystal formation and,


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respectively, to maximum crystal growth. “X” denotes no protein crystal formation was observed. Lipid

Time for microcrystal formation

Max. crystal size reached

7:7

< 24 hrs

3 days

MP

< 24 hrs

8 days

MO

≈ 48 hrs

11 days

MV

≈ 48 hrs

12 days

ME

X

X

PT

X

X

Table 3. Single crystal X-ray diffraction structural data for the intimin crystals obtained from various lipidic mesophases. The data shown for the MO-grown* crystals was independently obtained by the Buchanan lab PDB code 4E1S40 and used here for comparison with our diffraction data. “X” denotes no protein crystal formation was observed. The data for the MP-grown† crystals was collected during a previous study.38 Lipid

Resolution

Space group

Completeness

Cell size

7:7

3.74 Å

C2221

99.1 (97.0)

108.2 x 119.0 x 39.2 Å

MP†

2.42 Å

C2221

99.1 (91.8)

115.9 x 119.9 x 39.0 Å

MO*

1.85 Å

C2221

93.6 (71.5)

116.6 x 120.5 x 39.1 Å

MV

3.8 Å

C2221

98.9 (96.3)

107.9 x 118.9 x 39.2 Å

ME

X

X

X

X

PT

X

X

X

X

DISCUSSION: Interplay between bilayer thickness and protein/solute diffusion rates


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The first step in the LCP-based crystallization process lies with the empirical selection of the host lipidic mesophase. A previous study utilizing a series of unsaturated MAGs and the membrane porin, OprB, emphasized the importance of utilising a large library of lipids for such crystallization.58 Changes of as little as a single carbon in the length of the aliphatic tail (C15 vs. C14) were sufficient to promote or prevent crystallogenesis.58 Misquitta et al. further emphasize the possibility of using various cubic phase forming lipids by successfully crystallizing two robust membrane proteins, bacteriorhodopsin and BtuB, from MO and 7.7 MAG.59 The goal of our study was to explore how the chemical structure of the host lipids correlates with bilayer properties and, in turn, protein crystallogenesis. SAXS analysis revealed minimal structural changes subsequent to protein addition and provided information regarding mesophase geometry. The first clear indication of interplay between host lipid identity and bilayer properties was obtained by comparing calculated bilayer thickness with NMR determined lipid diffusion coefficients. Among MAG lipids, translational diffusion of lipid molecules within the hydrophobic bilayer was found to be directly dependant on the aliphatic chain length. More precisely, we noted an inverse correlation between the bilayer thickness of the phase and the lipid diffusion coefficient, with the highest diffusion coefficients being observed for the shorter chained lipids. Lipid diffusion within the PT system was, in some cases, nearly one order of magnitude lower than in MAGs, which we suggest is due to the highly branched and saturated nature of the PT hydrocarbon chain that influences lipid packing, as well as the significantly increased curvature and strain in the PT bilayers. The diffusion of intimin measured via FRAP followed the same trends as observed for lipid diffusion, showing a clear, inverse correlation between bilayer thickness and protein diffusion coefficient. Protein diffusion rates were considerably faster for the shorter chain MAGs,


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whereas molecular transport was again significantly slower in PT samples. The measured values for the protein diffusion coefficients correspond remarkably well to their lipid counterparts when bearing in mind the difference in molecular weight between the molecules and in experimental methods. Effect of protein/solute diffusion rates on protein crystal growth The increased lipid and protein mobility within the shorter chain lipid systems was directly correlated with faster nucleation times and crystal growth. Small crystals were observed after approximately 24 hours in the shorter chain MP cubic phase, whereas MO and MV required a minimum of 48 hours before the first crystals were observed. Moreover, the protein crystals reached their maximum size after 8 days in MP as opposed to 11 days for MO and 12 days for MV. A large mismatch between the size of the protein hydrophobic domain (estimated 24.4 Å)60 and the thickness of the lipid bilayer could potentially further influence protein mobility and, implicitly, nucleation and crystal growth. We propose that in the ME LCP, the larger hydrophobic mismatch between the bilayer thickness and the size of the intimin transmembrane domain, coupled with slower translational diffusion within the bilayer, resulted in a complete breakdown of crystallogenesis. Conversely, the fast diffusion of molecules in the 7.7 MAG sponge phase resulted in much faster nucleation and crystallization times: first crystals were observed after less than 24 hours and only required 3 days to reach their maximum size. In addition to the diffusion of protein molecules within the bilayer, crystal growth could also be driven by the diffusion of hydrophilic salts and crystallization agents (i.e. PEG 400) through the mesophase aqueous channels. In recent studies, a direct correlation was found between aqueous channel diameter and the diffusion rate of small molecules solvated within the water channel network of LCPs.55 Thus in addition to increased protein and lipid


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diffusion, the large water channel diameter of the MP mesophase likely also confers a crystallization advantage. The different molecular transport rates affect not only the kinetics of crystal growth but also the morphology of the formed protein crystal. Four distinct crystal morphologies were obtained from the shorter chain MAGs, from thick rectangular crystals in the case of 7.7 MAG to star-shaped clusters of thin plates in the case of MV. In spite of their appearance, all crystals belonged to the same crystallographic space group (C2221) and had remarkably similar unit cell sizes. These findings clearly indicate that the hosting cubic phase acts as a conduit of protein molecules capable of regulating the kinetics and macromolecular assembly of the protein molecules. Therefore, we have revealed the direct interplay between the host cubic phase properties and the process of nucleation and protein crystal growth. The ability of the cubic phase to transport membrane protein molecules appears to be directly controlled by the hydrocarbon chain length of the lipid molecules. This, in turn, dictates the time required by the protein molecules to form the necessary nuclei, develop crystals and reach maximum growth. Moreover, the bilayer-controlled transport properties of the cubic phase may well play a direct role in shaping the final morphology of the protein crystal which opens the door towards the possibility of engineering protein crystals at the molecular level. CONCLUSIONS: Having previously shown the mechanistic importance of a localized lamellar phase to the nucleation of membrane protein crystals,38 we have now identified a number of quantitative bilayer properties which significantly impact successful LCP-based protein crystallogenesis. Lipid and protein diffusion, measured using PFG-NMR and FRAP, respectively, were fastest in the MP-based mesophase which also yielded larger crystals in a shorter overall timeframe.


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In spite of distinct differences in kinetics, crystal size and morphology, the packing of the protein molecules and the space group of the obtained crystals were identical for crystals obtained from MO, MP, MV and 7.7 MAG cubic phases. The mesophases with the lowest measured translational diffusion coefficients, PT and ME, failed to yield protein crystals. Hydrophobic matching and aqueous channel nanoscale diameter were identified as factors contributing to successful crystallogenesis. These results provide compelling evidence for a direct interplay between LCP-based protein crystallization and molecular diffusion within the lipidic cubic phase. This in turn opens new avenues for the future design of rational and efficient systems for membrane protein crystallization. SUPPORTING INFORMATION: Supporting information is available, containing detailed intimin expression and purification methodology, the method for LCP structural parameter calculation, 1D SAXS plots for each sample listed in Table 1, full list of screen conditions used in crystallization experiments, FRAP recovery curves, and a diagram of intimin molecule packing within LCP-grown crystals.

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TOC Graphic:

Synopsis: A detailed, systematic investigation into the factors contributing to successful crystallization of membrane proteins in meso. We find compelling evidence for the role of the chemical structure of the lipid, the physical properties of the mesophase, and the translational diffusion of the encapsulated membrane protein itself.


27


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Figure 1. Plot of lipid diffusion coefficients determined experimentally using PFG-NMR versus bilayer thickness, calculated from SAXS measurements of pure lipid excess water mesophase systems. 208x213mm (300 x 300 DPI)


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Figure 2. Plot of experimentally determined diffusion coefficients of intimin reconstituted in a series of MAG lipids and phytantriol versus mesophase bilayer thickness, calculated from SAXS measurements of pure lipid excess water mesophase systems. Error bars indicate ± 1 SD. 208x213mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 3. Overview of LCP-based crystallization setups of intimin using the following LCPs: 7.7 MAG, MP, MO, MV, PT and ME. Images were obtained using an automated Minstrel HT/UV imaging system (Rigaku). In the case of ME, the observed aggregation and crystal formation in the drop is a result of lipid crystallization, with none of the formed crystals showing UV absorbance. 643x1018mm (150 x 150 DPI)


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TOC graphic 430x245mm (59 x 59 DPI)


Lipidic Cubic Phase-Induced Membrane Protein Crystallization

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