Or: Marcus’ summary of a brilliant but not yet fully understood advance in protein crystallography

1. A lipid’s eye view of membrane protein crystallization in mesophasesa journal club talk encompassing far too many papers, and borrowing a title from Caffrey, Current Op. In Structural Biology, 2000, 10:486-497 Or: Marcus’ summary of a brilliant but not yet fully understood advance in protein crystallography

2. Why do membrane proteins give us headaches? • The key problem is that membrane proteins like to be in membranes! They have both hydrophilic and hydrophobic regions! • An early approach was to solubilize membrane proteins in non-ionic detergents. • But why not solubilize them in membranes? This is the essential premise behind Landau & Rosenbusch, PNAS, 1996

3. the method

4. Lipid Liquid Crystals • Lipids form a variety of crystalline phases, including cubic, hexagonal and lamellar liquid crystals, in which the individual lipid molecules are relatively mobile. • Parameters such as salt concentration, temperature, lipid composition, and water concentration affect the lattice parameter and phase of these liquid crystals.

5. So? Does it work? • Short answer: yes. • Crystals of bR, hR, photosynthetic centers from Rcvir, RCsph, and LH2, and also soluble proteins have been refined to ~2 angstroms. • With the exception of RCvir, none of the MPs have extramembraneous regions, but the structures are sll somewhat different.

6. Why does this work? • In short, there is no good answer to this question. • Lateral pressure as a stabilizing mechanism? • “Salting out” vs. phase & lattice parameter effects. • “Feeding” of a nucleated crystal? • It is not fully known how the protein gets into the lipid in the first place.

7. Phase diagrams of MO systems

8. Caffrey’s view of the process A-B. detergent solubilizes membrane protein by surrounding hydrophobic regions. This is, however, very unstable. C. water begins to soak into MO Lc phase. D. some water farther into MO leads to cubic phases, while high detergent concentrations (~0.1M) in other regions leads to lamellar MO.

9. Caffrey’s view, part 2. E. protein is taken up in familiar, comfy, lamellar bilayer. F. as detergent diffuses, MO returns to a cubic phase G. addition of salt causes a disturbance, leads to lamellar phases, areas of decreasing cubic lattice parameter H. lamellar sheets feed a growing crystal

10. Problems • Salt concentrations are roughly 4M (a generous estimate, mostly made up of buffers), but Takahashi et al., Mol. Cryst. And Liq. Cryst. 2000, 347:231-8 suggests that the MO could remain cubic in up to 4M salt. • No actual nucleation mechanism is mentioned. This seems to be a critical point.

11. Other observations • The habit of the membrane protein crystals is strongly dependent on the medium in which it is grown • Another recent study (Essen, et al., PNAS 1998, 95:11-21) grew bR crystals epitaxially on benzamidine sulfate, with yet another space group for the bR crystals • Twinning has been observed in many bR crystals, as well as high mosaicity (a number is not given) in 9 out of 10 of ELM’s samples. • Proteins with extramembraneous regions larger than the water channels in the cubic phase have been incorporated into and crystallized from these phases.

12. All of which leads Marcus to believe that • The lamellar phase is probably not necessary • The role of the lipid is probably two-part: first to provide a stable and native environment that the protein can survive in, and second as a substrate. • In this substrate, the protein diffuses gradually, even forming small groupings of “protocrystals”, a few mers each, and which are probably quite unstable. Notably, bR naturally forms 2D crystals in bilayers. • Once “salt” is added, the lattice parameter will decrease, forcing protocrystals out of the lipid proper. At this point, the protocrystals probably are quite stable, given that they don’t have anywhere to go. From here, the lipid still shields the baby protein crystal, and provides a deformable substrate in which protein monomers can diffuse easily, eventually attaching to the growing crystal.