A lipids eye view of membrane protein crystallization in mesophases a journal club talk encompassing

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A lipids 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, 10486-497
Or Marcus summary of a brilliant but not yet
fully understood advance in protein
crystallography
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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

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the method
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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.

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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.

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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.

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Phase diagrams of MO systems
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Caffreys 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.
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Caffreys 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
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Problems

• Salt concentrations are roughly 4M (a generous
  estimate, mostly made up of buffers), but
  Takahashi et al., Mol. Cryst. And Liq. Cryst.
  2000, 347231-8 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.

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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,
  9511-21) 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 ELMs samples.
• Proteins with extramembraneous regions larger
  than the water channels in the cubic phase have
  been incorporated into and crystallized from
  these phases.

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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 dont
  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.