1. Refolding of membrane proteins for structural studies Lars Linden * RAMC 2005

2. Membrane proteins as drug targets m-phasys is the only company focussed exclusively on 3D structures of membrane protein targets The human genome:  25% of the human genes encode for membrane proteins 75%25% membrane proteins soluble proteins  67% of the known drug targets are membrane proteins The known drug targets: 67%33%

3. All known protein structures: ~ 28,000 No human GPCR structure solved GPCR structures: 1 (Rhodopsin from bovine retina) Membrane protein structures: ~ 100 (mostly bacterial proteins) Why ? Human GPCR structures: 0 PDB

4. Barriers in membrane protein structural analysis...... and how to get around them Expression system Purification & Crystallization 3D structureDNA Refolded protein Expression in Inclu- sion bodies Detergent Solubilized protein crystal

5. E. coli ? Fast Cheap High yields Multiple strains available Multiple plasmids available Selenomethionine derivatives Less time for expression = more time for crystallization! In 2004, 67% of all structures deposited in the PDB were from proteins expressed in E. coli

6. Percentage of structures from proteins produced in E. coli

7. Expression of membrane proteins in E. coli can be toxic Eukaryotic membrane proteins are not readily inserted into bacterial membranes Bacterial insertion machinery becomes jammed Protein production stops after 1 min Low yields  Possible solution: Prevent membrane insertion

8. Does in vitro refolding of membrane proteins work ? Critical issues: Energy landscape in micelles? Non-vectorial insertion Local vs. Global minimum?

9. Does in vitro refolding of membrane proteins work ? Yes! Bacteriorhodopsin Light harvesting complex LHC2 Mitochondrial transporters Diacyl glycerol kinase Olfactory receptor OR5 Potassium channel KcsA DsbB Leukotriene receptor BLT1

10. pGEX2a-GPCR-His ~6000 bp AP r GST lac I HisTag Ptac ori rrBT1T2 Protease cleavage site GPCR Expression vector for GST-GPCR- (His)6 fusions Expression in E.coli Preparation of inclusion bodies Typical yields: 2-50 mg / l

11. How to identify refolding conditions Inclusion Bodies (Aggregated Protein) Refolded & nativeMisfoldedRe-aggregated Solubilisation Solubilised, but misfolded protein Detergent exchange

12. Purification and quality control of GPCRs Principal analysisThreshold Purity (SDS-PAGE):> 90% Monodispersity (SEC)> 90% Specific activity (arrestin assay*) > 70% Concomitant analysis Light scattering (DLS) Ligand binding measurement G protein activation GPCRs are rigorously tested for activity and homogeneity before crystallization *) proprietary functional assay applicable to all GPCRs (including orphans)

13. Arrestin activity assay Arrestin mutant binds to GPCRs constitutively Doesn't require phosphorylation Affinity depends on ligand binding Requires folded GPCR R A A R A R A A R 1. Bind & wash 2. Detect bound arrestin  GPCR properly folded  GPCR not properly folded

14. G protein activation Log Interleukin-8 [M] Bound GTPS [dpm] Ligand binding Log Interleukin-8 [M] Bound ligand [dpm] Refolded GPCRs are functional Example: CXCR1 Refolded GPCR binds ligand and couples to G protein Conclusion: Ligand affinity (K D ) like native receptor > 80% refolded (B max ) Conclusion: Couples to G i/o EC 50 like native receptor

15. Refolded GPCRs are homogenous Example: CXCR1 SDS-PAGE 1 2 3 - GPCR dimer - GST-GPCR fusion - GPCR monomer 1.Inclusion body fraction 2.Ni chelate purified 3.SEC purified Refolded CXCR1 is >90% pure and monodisperse Conclusion: 95 % pure on SDS gel Conclusion: 85 % pure by SEC analysis SEC Absorption Volume [ml] Superdex 200

16. Refolded GPCRs are homogenous Example: GPR3 AnalysisResult Purity (SDS-PAGE):95 % Monodispersity (SEC)90 % Specific activity (arrestin assay) 80 % Absorption Volume [ml]

17. Refolded GPCRs form crystals Crystallized Pipeline Crystallized Pipeline Rhodopsin family    

18. Optimization of crystallization conditions: strategy Truncated mutants (N- and C-termini, long loops) Co-crystallization with ligands (agonists, antagonists, inverse agonists) Co-crystallization with binding proteins (ß-arrestin, G proteins, antibody fragments) Stabilization with lipids Variation of crystallization method: vapour diffusion, microbatch, lipidic cubic phases, free interface diffusion Selection for more thermostable mutants

19. Anti-GPCR monoclonal antibodies Successful programs with antibody companies and academic groups Refolded GPCRs used as immunogen or panning target Antibodies obtained from mice (IgG) and phage display systems (scFvs and Fabs) Antibodies recognize native GPCRs (FACS) Affinity from 1 nM to 1 µM Some are antagonistic Some have conformation-specific epitopes  Apart from their use in co-crystallization, antibodies might be used as diagnostic tools or therapeutics

20. m-fold CXCR1-antibody complex formation Immunization with CXCR1 Liposomes Monoclonal IgG, FACS and ELISA positiv Ligand (IL-8) is displaced by antibody (IC 50 = 0,33 nM) CXCR1 receptor and 9D1 antibody form a stable complex scFv cloned, expressed and purified -> Co-crystallisation

21. Bacterial and human ion channels Potassium Channels : voltage gatedKvLQT4 hERG Kv1.3 VIC (Salmonella t.) MJKch (Methanococcus j.) Ca 2+ activated KCa 4 Cloning and expression of different constructs of hERG, Kv1.3, KCa 4 transmembrane region S1-S6

22. Bacterial and human ion channels Ion channels are easily purified Refoldung screen for hERG, Kv1.3, KCa 4, VIC and MJKch Tetramerisation can be detected on modified SDS or blue native Gels VIC tetramer monomer hERG tetramer 66 132 116 66 45 35 25 116 66 45 35 25 18

23. Potassium channel can be produced with M-FOLD™ Refolding works for K channels 116 66 45 35 25 18 unfolded 116 66 45 35 25 18 refolded Conclusion: Refolded K channel forms tetramer > 95 % refolded Refolded K channels reconstituted into planar bilayer (BLM)

24. K channel crystals Ion channel crystals diffract to 12 Å

25. Acknowledgement