1. Membrane Proteins: The Next Structural Challenge For the average eukaryotic genome, 20-30 % of all proteins are membrane bound and serving essential functions. Qin et al. P.N.A.S. 103(44) 16117-16122 For the average eukaryotic genome, 20-30 % of all proteins are membrane bound and serving essential functions. Barber p. 619

2. Membrane Dynamics – Membrane Anchors

4. Different types of anchors may also be associated with inner versus outer leaflet association.

5. Membrane Anchors Intergral membrane proteins may also contain anchored regions.

6. Traditional Membrane Protein Isolation

7. Crystallization in Cubo

9. Crystallization in Cubo (cont.)

10. In Cubo Techniques Capture Native Lipids Taken from Luecke et al. JMB 291 p. 899-911 PDB 1C3W

18. The Sponge Phase Wadsten et al.

19. The Sponge Phase (cont.) Transformation of the LCP to the sponge phase monitored by SAXS.

20. The Sponge Phase (cont.) Crystals of the reaction center from Rhodobacter sphaeroides

21. The Sponge Phase (cont.) Quinone A and B are critical to function (electron flow and proton pumping). More importantly crystals could be grown directly in the “sponge” phase thereby bypassing any symmetry constraints associated with the LCP.

22. Antibodies as Tools for Crystallization Hunte and Harmut (2006) in Membranes p. 503-508

23. Antibodies as Tools for Crystallization (cont.)

24. Fab is a monoclonal antibody that ony recognizes tetrameric form of the channel.

25. Replacing the detergent with cyclodextrin, ring-shaped molecules made up of 6, 7, or 8 molecules of glucose.

26. General structure of a Cyclodextrin Ring

27. 2D Crystallization (cont.) Cyclodextrin “hides” detergent forcing membrane protein molecules to interact.

28. 2D Crystallization (cont.)

30. Learning More About Lipids High resolution structure obtain by engineering out molecular heterogeneity.

31. Learning More About Lipids (cont.)

32. PC, phosphatidyl choline; PG, phosphatidyl glycerol; CDL, cardiolipin; TGL, triacylglycerol Conserved lipid binding sites provide membrane orientation markers and functional clues.

33. Learning More About Lipids (cont.)

34. Conserved water positions provide proton transport chains.

35. Conclusions At the present time, the purification and crystallization of membrane protein both require information about membrane composition. A number of techniques exist that may be successful in both cases. - Detergent solubilization - Cubic Lipid Phase - Sponge Phase - Antibody co-crystallization Further basic research must be done to establish the organization and composition of membranes. Why? The purification and crystallization both require screening a vast array of lipids/detergents. This matrix becomes exponentially more complex when one must also consider the library of precipitants that need to also be screened during crystallization.