Production and crystallization of membrane proteins. So Iwata Imperial College London / Diamond Light Source

Transcription

1 Production and crystallization of membrane proteins So Iwata Imperial College London / Diamond Light Source

2 Membrane Proteins: the Last Frontier in Structural Biology Only ~190 unique membrane protein structures Less than 10 unique mammalian membrane protein structures Coordinates in Protein Data Bank (PDB) Soluble Proteins ( ) Membrane proteins from prokaryotes Membrane proteins from eukaryotes 60, ,000 40, , ,000 10, Year Year

3 Membrane Transporters: a Major Class of Membrane Proteins Second largest family among membrane proteins 5-12% of genes in genomes encode membrane transporters Receptors GPCRs Unknown Transporters Channels Other enzymes Other membrane receptors Others Nuclear receptors Channels Human membrane proteins Transporters Drug targets

4 Solute Carrier Family (SLC) Transporters Ions (H +, Na +...) Membrane transporters including over 300 members organized into 47 families (by Human Genome Organization) Composed of facilitative transporters (passive transporters) and secondary active transporters (use an ion gradient for energy). Substrate Outwardfacing cavity Outside Ion pumps Membrane SLC Outward-facing conformation Inward-facing cavity Substrateoccluded conformation Inward-facing conformation Inside Ions (H +, Na +...)

5 Previous Work Experience in solving diverse membrane protein structures. Technical development for production and crystallisation of membrane proteins. Establish Diamond-Membrane Protein Laboratory (MPL) for high-throughput crystallisation of membrane proteins and high-quality data collection from membrane protein crystals. Oxygen Respiratory Complexes Complex III Anaerobic Respiratory Complexes Formate Dehydrogenase Complex II Complex IV Complex II: Science Complex III: Science 281, 1998, N. Engl. J. Med. 341,1999 Complex IV: Nature 376, 1995, Nature Str. Biol. 7, 2000 Nitrate Reductase F o r m a t e D e h y d r o - genase: Science Nitrate Reductase: Structure 12, 2004 Polysulfide reductase: Nat. Str. Mol. Biol. 15, 2008 Photosynthesis Transporters Photosystem II LacY Mhp1 P h o t o s y s t e m I I : Science LacY: Science 301, 2003, EMBO J. 25, 2006, PNAS 104, 2007 Mhp1: Science 322, 2008

6 SLC Crystallography Pipeline To develop methods to address major bottlenecks in membrane protein crystallography and to establish a pipeline for solving SLC structures To solve structures of 5 SLC families Key parameters to monitor Activity Expression level Integrity Monodispersity Thermal stability Detergent concentration Resolution Indexing Scaling and merging Processes Expression ~200 Purification ~50 Crystallisation ~14 Data collection ~6 Structure determination? Technical options/ developments Construct design Expression systems Culture conditions Detergent choice Purification methods Additives Crystallisation methods Detergent choice Additives Antibody Data collection systems Sample mounting Phasing methods Model building and refinement

7 GFP-based Membrane Protein Expression Systems in S. cerevisiae Fur4 Azr1 AGP1 Isc1 Hsp30 Sec61 YOL162W ITR1 Shr3p HNM1 HXT3 HXT1 UGA4 MPH3 ARN1 DAL4 TPO4 DUR3 KHA1 Rft1 SsH1 GAL

8 GFP-based Membrane Protein Purification Systems in S. cerevisiae (and E. coli) MP-TEV-GFP-8His 1. S c r e e n membrane protein- G F P o v e r - e x p r e s s i o n b y w h o l e - c e l l f l u o - rescence 2. Isolate crude membranes N 1.Detergentsolubilisation GFP 8His C TEV10His 2.Incubation of supernatant with Ni-NTA resin Drew et. al. Nat. Methods. 2, 2006 Newstead et. al. PNAS 104, 2007 Drew et al., Nat. Protoc. 3, I M A C purification 3.Check integrity u s i n g i n - g e l fluorescence Ni-NTA resin 4. Check cellular localisation by confocal microscopy 5.Check monod i s p e r i s t y i n different detergent by fluorescence s i z e - e x c l u s i o n chromatography Spin down nondetergent solubilized proteins 4. D i a l y s i s and cleavage of GFP-8His 5. R e v e r s e IMAC to remove GFP-8His and his-tagged TEV Concentration of membrane protein Flow-through Wash Elution 6.Gel filtration of membrane protein (option)

9 Protein integrity test using in-gel fluorescence

10 Cellular localisation test by confocal micro-scopy CONFOCOL WIDE-FIELD ER protein ER transport protein Rft1 Plasma membrane protein Plasma memb rane ctr1 Golgi transport protein Golgi vrg4

11 Monodisperisty test in different detergent by fluorescence size-exclusion chromatography (1) Kawate, T, Gouaux, E (2006). Structure

12 Monodisperisty testin different detergent by fluorescence size-exclusion chromatography (2) Use less than 1% of sample to screen detergent

13 Purification of a GFP-fusion protein Non-solubilized membranes Solubilized membranes Flow-through Ni-NTA bound LacY-GFP LacY-GFP eluate LacY-GFP GFP / TEV LacY LacY-GFP GFP / TEV LacY LacY-GFP GFP / TEV LacY Natural light In-gel fluorescence Coomassie stained

14 Crystallisation of Membrane Proteins Large micelle detergents stabilise membrane proteins Small micelle detergents suitable for crystallisation Antibody fragment facilitates membrane protein crystallisation even in large micelle detergents Cytochrome c oxidase Antibody Fv fragment Membrane protein Using smaller detergent Membrane protein crystals Antibody Fab or Fv fragment Detergent micelle Extend polar surface using antibody fragment Crystals of membrane protein - antibody complex Cytochome c oxidase - antibody Fv fragment crystal (Iwata et al. Nature 376, 1995). 14

15 Systematic Optimisation Strategy for Transporter Crystals Based on Stability Assay Improve crystal resolution beyond 4.0 Å to solve structures, if possible, beyond 2.5 Å for good structures. The CPM assay was developed to help select a suitable small detergent. The CPM assay can also be used to select additives to stabilise target. Principle of the CPM assay folded Cys CPM Detergent Incubate with CPM at 40 deg unfolded Fluorescence is enhanced CPM: the thiol-specific fluorochrome, N-[4-(7-diethylamino-4-methyl-3- coumarinyl)phenyl]maleimide Effect of detergents Stability - Crystallisation + Stability + Crystallisation - Effect of lipids Stability + Crystallisation +

16 Crystallization of Membrane Proteins using Antibody Fragment or Artificial Binder Antibody or Binder Detergent Micelle Membrane Protein Detergent solubilised membrane proteins (difficult to crystallize b e c a u s e o f t h e detergent micelle). M e m b r a n e protein Antibody/ binder complex C r y s t a l o f t h e membrane protein - a n t i b o d y / b i n d e r complex

17 Plan of the MPL Facility A u t o m a t e d C r y s t a l Optimisation at Diamond MPL Fully integrated sitting-drop Rhombix crystallisation robot Liquid Handler Robotic arm for plate movement Fluidigm Toparz microfluidics system Nanodrop crystallisation robot Cubic phase crystallization robot Crystal hotel/imager Remote access database and control system PX scanner in-plate X- ray diffraction system High-throughput crystallisation/ optimisation of SLC crystals will be performed at Diamond- MPL Diamond-MPL Funded by Wellcome Trust/ Japan Science and Technology Agency/BBSRC Imperial/Diamond joint project Methods development for automated membrane protein crystallisation and high quality X-ray data collection User training and research facility for membrane protein c r y s t a l l i s a t i o n a n d d a t a collection

18 Integrated Crystallisation System: Plate preparation Drop setting Plate storage Plate imaging Data storage

19 Fully Automated Protein Crystallisation at MPL Prepare crystallisation solutions in 96 well plates Transfer to nanodrop robot S e t n a n o d r o p s, transfer to sealer Transfer to 20ºC or 4ºC imager MemGold : Molecular Dimensions 113 unique alpha-helical membrane protein structures Newstead, S, Ferrandon, S, Iwata, S Protein Science (2007). C r y s t a l observation u s i n g d a t a b a s e software

20 Reagent chamber Protein chamber Contain ment Valve Interf ace Valve 1.5 mm Contain ment Valve

21 Bacteriorhodopsin and related proteins GPCRs Caffrey, Acta Cryst D 2004

22 UV Microscope Molecular dimensions Excites around nm Absorbs around 250 nm Swissci plates Caffrey glass plates IWATA HUMAN RECEPTOR CRYSTALLOGRAPHY PROJECT AT KYOTO

23 Small X-ray generator Takes 96 well plates only Good for eliminating salt Strong lysozyme diffraction 1 hour shot gives something with better membrane protein crystals

24 X-ray Data Collection at the Diamond Microfocus Beamline I24 Beamline I24 at Diamond. A. Beamline optics. B. Beam focus options. C. Sample environment with Irelec CATS robotic sample changer, grid scanning system (left) and the plate gripper (right). D. Aperture array. M e m b r a n e p r o t e i n crystals are often weakly diffracting, small and radiation sensitive. Diamond beamline I24 is designed to collect high quality data from micro crystals (5-50 µm). D e v e l o p i n g a n e w crystal mounting system a n d s o f t w a r e t o complete a dataset from multiple crystals to avoid radiation damage. Collaboration with Dr. Evans, I24 principal beamline scientist

25 MePNet E-MEP MPSI Imperial College London Centre for Structural Biology ERATO Kyoto Lab Imperial Diamond MPL Riken Yokohama Institute Diamond London Diamond-MPL ERATO-Kyoto Lab