Novel aspects of lipidic cubic phase-based crystallization. Linda Johansson University of Gothenburg, Sweden

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1 Novel aspects of lipidic cubic phase-based crystallization Linda Johansson University of Gothenburg, Sweden

2 Who am I? Has worked on membrane protein crystallography and lipidic phases since 2007 Is a Ph.D. Student in Richard Neutze s group at the University of Gothenburg My research focuses on nanocrystallography using lipidic sponge phases for Free-electron Laser experiments Also macromolecular crystallography using lipid-based phases (incl. LCP and bicelle).

3 Lectures outline Part I: Lipidic cubic phase Part II: Lipidic sponge phase Bicelle crystallization

4 Part I: Lipidic Cubic Phase for membrane protein crystallography- History, practical advice and successful examples

5 Membrane proteins Need to be cloned, overproduced and purified before crystallization. Difficult to crystallize Conventional crystallization approach based on detergent micelles Other methods include crystallization in oil, or stabilization using a lipid bilayer

6 The Lipidic Cubic Phase (LCP) Is a protein/lipid mixture used mainly for crystallization of membrane proteins Suitable for most types of membrane proteins Those containing cofactors (esp. hydrophobic ones) might not be suitable Standard techniques such as robots (Mosquito), additive screens, hanging and sitting drop are applicable. If the lipid MO is used, the crystallization must occur at room temperature

7 The Lipidic Cubic Phase (LCP) First membrane protein crystallized was bacteriorhodopsin (br) by Landau & Rosenbusch in 1996 Rhodopsins Photosynthetic proteins (RC, LHII) Several GPCRs br crystals in: a) MO (MAG 9.9) b) MP

8 Main ingredient: Lipid The phase requires a monoacylglycerol (MAG), with one unsaturated bond Most commonly used is Monoolein (MO), which is a MAG 9.9 Monopalmitolein (MAG 9.7) Monovaccenin (11.7 MAG) MAG 7.7, and MAG 7.9 Monovaccenin Unsaturated cis-bond Monoolein

9 Main ingredient: Lipid Lipids used often have Carbons in their chains MO is not stable below 17 degrees (forms a lamellar phase), i.e. the crystallization must occur at room temperature Shorter MAGs can be used at lower temperatures A 7.7 MAG can be used at 6 C Monovaccenin Ester bond

10 Monoolein (MO), MAG 9.9 Most commonly used A MO cubic phase can host various other agents: 20 mol% DOPC, DOPE 25 mol% cholesterol (esp. Eukaryotic proteins) 5 mol% DOPS 7mol% cardiolipin (esp. Prokaryotic proteins) Cardiolipin can be used in conjunction with MO to increase stability of proteins during crystallization. Mostly used for proteins of prokaryotic origin.

11 Properties of the LCP Semi-solid, toothpaste. a 60:40 mixture of lipid:protein, forms spontaneously Several space groups are possible. Stable at room temperature, MO will revert to lamellar liquid crystalline phases if below 17 C. The phase diagram of water and MO. Hydration limit occurs at approx. 40% (w/w) water at 20 C Cherezov et al. (2006). JMB 357:

12 Properties of the LCP Compatible with most other commonly used crystallization agents Additives (screens) Precipitants Pore size will be affected by the chain length and degree of swelling Protein To the right, the protein is inserted into the bilayer with one side facing the water channel of the LCP.

13 Crystallization path Protein molecules migrate and cluster Proposed to be a local change from cubic to lamellar phase Dehydration needed 2D stacked crystals often form, which usually diffract well Cubic Lamellar Caffrey & Cherezov (2009). Nat Prot 4:706-31

14 Crystallization using LCP Several ways of preparing the phase Sonication (of all components) Mixing in syringes Robot plates containing MO, overlaid with buffer Protein and lipid is mixed in a 60:40 ratio and precipitant is added

15 Requirements: manual setups Crystallization-grade protein Protein conc preferably higher than 10 mg/ml Lipid of choice (MO or other) Precipitant solutions Two gastight syringes with couplers Crystallization plates (glass or plastic) Cover slides or tape for sealing MiTeGen loops for harvesting

16 Manual setups 1. Decide on lipid composition (if a second lipid is to be added, prepare this the day before the crystallization experiment) 2. Prepare the precipitant solutions 3. Make sure that you have all the tools and chemicals ready before preparing the LCP 4. Mix the lipid and the protein in syringes 5. Dispense in a crystallization tray 6. Overlay with precipitant solution 7. Seal the crystallization plates 8. Incubate at Inspect the plates on a regular basis, e.g., day 0, 1, 2, 5, 7 and so on

17 Precipitant solutions The first LCP conditions for br did not make use of any precipitant solutions, only Na/K buffer or Cl - Common precipitants include MPD, Jeffamine, PEGs of various MW, PPO, ethanol All of these affect the phase through either swelling or shrinking (ethanol)

18 Stabilizing agents Various agents can be added for stabilization (either to the protein or to the phase) Antibodies Nanobodies Cholesterol Cardiolipin Glycerol Co-factors (such as quinols) Drugs, inhibitors, pesticides Cholesterol

19 Adding lipids to MO Weigh in the required amounts of the second lipid (typically 10-20%) along with MO (80-90%) Dissolve the lipids in a 2:1 mixture of chloroform and methanol Remove the solvent by a nitrogen stream Remove the last traces of solvent using vacuum (over night)

20 Destabilizing agents and actions Low (below 5) and high ph (above 9): the ester bond in the lipid will hydrolyse High temperature will also hydrolyse the lipid (and damage the protein) Excess of detergent can destroy the LCP Storing the MO in room temperature is not recommended MO from Nu-Chek Prep should be stored in the freezer

21 Practical advice: overview Requirements Experimental setup Optimization Finding and harvesting crystals Troubleshooting The LCP kit from Emerald Biosystems.

22 Practical advice: choice of lipid The size of your protein, protruding parts? MO easily avaliable, but others need to be synthesized Temperature? If your protein is not stable at RT, try shorter MAGs MO, which has a fairly long tail (MAG 9.9, total of 18 carbons)

23 Practical advice: filling syringes Use two gastight Hamilton syringes ( µl) Fill one syringe with molten lipid (40%) using a pipette with a cut off pipette tip (MO density: g/cm 3 ), while the plunger is inserted Fill the other with protein (60 %) Avoid air bubbles! Attach the syringes to each other using a coupler

24 Practical advice: mixing Push the contents of one syringe into the next Mix until the phase is transparent (NOT cloudy) Coupler Contents on one side!

25 Practical advice: crystallization setups Crystallization plates Red: Protein:Lipid mixture Blue: Precipitant Caffrey & Cherezov (2009). Nat Prot 4: Glass vials Protein and lipid is mixed, overlaid with precipitant and the vial is sealed

26 Practical advice: optimization One a hit is found, optimization will be the next step. Try first by optimizing: ph, salt (concentration), protein concentration, additives, bolus size, temperature, time Try seeding Change to other lipid is a big step! Seed beads from Hampton Research

27 Practical advice: finding crystals How does a protein crystal differ from its surroundings? Use common tools such as: Standard microscopy Polarization microscopy (birefringent crystals) Harvest crystals using a mesh loop and scoop up as much as you can. Use control plates (without protein)

28 Practical advice: harvesting Use of MiTeGen loops (including mesh loops) will ease the harvesting (the LCP is very viscous) MiTeGen loop Upon removal of cover slide, add a drop of the precipitant solution to avoid drying out. Often the lipid and precipitants act as cryo protectants crystals can be frozen directly

29 Troubleshooting: cloudy phase 1. Excess protein solution 2. Excess lipid 3. Air is trapped 4. Detergent conc. too high (lamellar phase) Cloudy?

30 Troubleshooting: leakage Inspect the syringes (incl. plungers, nuts etc) before Check that the connection is not loose The syringe could be broken The coupler might leak There could be something clogging in the system

31 Troubleshooting: mixing troubles The syringes and coupler will heat up when mixing Occasionally placing it on ice will hopefully reduce the heat and preserve the protein If the mixing needs a lot of force, the syringes or coupler are probably clogged.

32 Troubleshooting: dehydration Often occurs if the wells are not properly sealed or during harvesting Other phases are formed Overcome problems by avoiding exposure to the air Humidifier Cover the drop with precipitant solution when harvesting

33 Visualizing the drop: phase transitions a) Dried out mesophase b) Crystalline lamellar phase c) Lamellar phase d) Lipidic sponge phase e) Hexagonal phase f) Fatty acid hydrolysation Caffrey& Cherezov (2009). Nat Prot 4:

34 Visualizing the drop: promising results a) br crystals b) LHII crystals c) OpcA d) BtuB e) Human engineerd β2 adrenergic receptor-t4 lysozyme f) Pseudomonas carbohydrate transporter Caffrey & Cherezov (2009). Nat Prot 4:

35 Other problems Colorless proteins difficult to spot Chromophores might be lost Novel data collection methods and micro-focus beamlines have greatly improved the success rate. Possible to merge data from several small crystals Ubiquinol. Hydrodphobic cofactor often lost during LCP crystallization.

36 Commersial aspects: LCP Screens Jena Bioscience Hampton Research Emerald biosystems Molecular Dimensions Kit from Jena Bioscience Most screens are for use with robotics LCP-containing robot plate are avaliable (Qiagen)

37 Commersial aspects: Lipid avaliability MO avaliable from Nu-Chek prep inc, Sigma MV avaliable from Nu-Chek prep inc MP from Affymetrix, Nu-Chek prep inc Some are not commersially avaliable and must be synthesized. MO from Nu-Chek prep inc

38 Unique structures from LCP Protein Resolution (Å) Source PDB entry Lipid Year First lipidic phase structure Bacteriorhodopsin 2.50 H. salinarum 1AP9 MO,MP 1997 Pebay-Peyroula et al. Halorhodopsin 1.80 H. salinarum 1E12 MO 2000 Kolbe et al. Sensory rhodopsin II 2.10 N. pharaonis 1H68 MO 2001 Royant et al. Sensory rhodopsin II-transducer complex 1.94 N. pharaonis 1H2S MV 2002 Gordeliy et al. Reaction center 2.35 R. sphaeroides 1OGV MO 2003 Katona et al. Anabaena Sensory Rhodopsin 2.00 Anabaena 1XIO MO 2004 Vogeley et al. Engineered human β 2 adrenergic receptor 2.40 Homo sapiens 2RH1 MO 2007 Cherezov et al. OpcA outer membrane adhesin 1.95 N. meningitidis 2VDF MO 2007 Cherezov et al. Human A 2A adenosine receptor 2.60 Homo sapiens 3EML MO 2008 Jaakola et al. CXCR4 Chemokine receptor 2.50 Homo sapiens 3ODU MO 2010 Wu et al. Dopamin D3 receptor 3.15 Homo sapiens 3PBL MO 2010 Chien et al. ba 3 cytochrome c oxidase 1.80 T. thermophilus 3S8F MO 2011 Tiefenbrunn et al. Histamine H1 receptor 3.10 Homo sapiens 3RZE MO 2011 Shimamura et al. MO= Monoolein, MV= Monovaccenin, MP= Monopalmitolein The first bacteriorhodpsin crystals from LCP were discovered in 1996 by Landau and Rosenbusch, although this is not in the pdb!

39 Successful examples: Rhodopsins SRII with transducer using MAG 11.7 MDP as a precipitant. Halorhodopsin, MAG 9.9. Salt only. br has been crystallized using MAG 7.7 and MAG 9.7. No Precipitant, only salt

40 Successful examples: RCs Reaction center from R. sphaeroides grown from LCP in glass vials Lipid: 100% MO Precipitant: 18% Jeffamine M600 Incubated at 20 for 3 days Close- up. The purple crystals show up nicely. Katona et al JMB 331 :

41 Successful examples: ba 3 cytochrome c oxidase Lipid: 100% MO Precipitant: % PEG400 Incubated at 20 for 14 days Tiefenbrunn et al. (2011) PloS ONE 6(7): e22348

42 Successful examples: GPCRs The human histamine H 1 receptor complex Lipid: 10 % Cholesterol, 90% MO Precipitant: 30% PEG400 Incubated at 20 for 7-14 days CXCR4 Chemokine Receptor Lipid: 10 % Cholesterol, 90% MO Precipitant: 20% PEG400 Incubated at 20 for 10 days Shimamura et al Nature 475: Wu et al Science 330:

43 Successful examples: GPCRs β 2 -Adrenergic Receptor (bound to various agonists, antagonists, nanobody, potential drug) Lipid: 10 % Cholesterol, 90% MO Precipitant: 32% PEG400 Incubated at 20 for 3-7 days

44 Successful examples: GPCRs Adenosine A 2A Receptor Dopamine D3 Receptor Lipid: 10 % Cholesterol, 90% MO Precipitant: 30% PEG400 Incubated at 20 for days Lipid: 10 % Cholesterol, 90% MO Precipitant: 30% PEG400 Incubated at 20 for 14 days Jaakola et al. (2008) Science 322: Chien et al.(2010) Science 330:

45 Successful examples: GPCRs All published GPCR structures have some common themes. The LCP consists of 50 nl: 60% is lipid (10% Cholesterol and 90 % MO), 40% protein The precipitant solution (0.8 µl) contains % PEG400 Nanobodies, antibody fragments, agonists, ligands, drugs etc for stabilization of a conformational state Sponge phase conditions contain % PEG. Are these conditions in fact LSPs?

46 Recommended reading: Structures Landau & Rosenbusch., (1996) Lipidic cubic phases: a novel concept for the crystallization of membrane proteins Proc Natl Acad Sci U S A 93: Katona et al. (2003) Lipidic cubic phase crystal structure of the photosynthetic reaction centre from Rhodobacter sphaeroides at 2.35 Å resolution J Mol Biol 331: Cherezov et al., (2006) Room to move: crystallizing membrane proteins in swollen lipidic mesophases J Mol Biol 357: Cherezov et al., (2006) In meso structure of the cobalamin transporter, BtuB, at 1.95 Å resolution J Mol Biol 364: Cherezov et al., (2007) High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor Science 318: Jaakola et al,. (2008) The 2.6 Angstrom Crystal Structure of a Human A2A Adenosine Receptor Bound to an Antagonist Science 322: Shimamura et al.(2011) Structure of the human histamine H1 receptor complex with doxepin Nature 475: 65 70

47 Recommended reading: Reviews Johansson et al (2009). Membrane protein crystallization from lipidic phases. Curr Opin Struct Biol, 19: Caffrey & Cherezov (2009). Crystallizing membrane proteins using lipidic mesophases. Nature Protocols, 4: