1. Membrane Protein Crystallization From Cubic Lipid Matrices
Marcus D. Collins and Sol M. Gruner,
Cornell University LASSP
Membrane proteins at a glance.
Protein crystallography.
What makes membrane proteins hard to crystallize?
What has succeeded?
The open questions and our project.
The x-ray determined structure of the light harvesting protein bacteriorhodopsin, from
Halobacterium halobium, (5)

2. Membrane proteins at a glance
Membrane proteins are simply those that exist in cell membranes. They can serve as structural supports, as both passive and active channels for ions and chemicals, or serve more specialized functions such as light reception. Fully one quarter of the human genome encodes membrane protein sequences.
Proteins in a bilayer membrane patch, (1)
One of the defining features of membrane proteins is that both hydrophobic and hydrophilic regions exist on their surfaces. This allows the proteins to blend in to the hydrophobic region created by the lipid bilayer which makes up most of the membrane, and still to have a stable interface with the aqueous material on either side of the membrane.
Because of these environmental restrictions placed on membrane proteins, it seems likely that their structrural possibilities are more limited than aqueous proteins, though this is not fully explored. Common features include transmembrane a-helices arranged like the staves of a barrel, “sheet structures” called b-barrels, and sometimes large extramembraneous regions of hydrophilic amino acid residues.
Various membrane proteins shown from views parallel (above)
and perpendicular to the membrane, (6)

3. Protein crystallography
Just like minerals and salts, proteins can form crystals. And, just like other crystals, proteins will diffract X-rays. From the X-rays diffraction patterns, the protein structures can be determined.
bR crystals surrounded by lipids, (5)
However, proteins are much harder to grow than salts, and by 1975 only 37 structures had been placed in the Protein Data Bank. These crystals tend to be extremely fragile and sensitive to conditions such as pH, specific ion concentrations, and other factors. They are also easily damaged by the X-rays used to probe their structure.
X-ray diffraction images of bacteriorhodopsin crystals, (3)
Now, as techniques of growing crystals and inverting the X-ray scattering data have improved, more than 12,000 structures have been posted to the PDB.

4. What makes a membrane protein hard to crystallize?
Remember that membrane proteins have both hydrophilic and hydrophobic regions. Until very recently all protein crystallization techniques used an aqueous solvent for crystallization. Membrane proteins easily denature (that is, lose their structure) in this environment. In 1984 the first membrane protein, a photosynthetic reaction center, was crystallized and its structure determined, earning a German trio the Nobel Prize.
Detergent surrounds membrane proteins in solution, (1)
These early efforts centered around using a new class of synthetic, highly contrived detergents which surrounded the proteins and protected them-if just barely-from the nearby water.
Despite this advance, only a small number of membrane proteins have been crystallized to date, largely because no general procedure has been found which can crystallize a variety of membrane proteins. The process of finding the right detergent and the correct conditions for crystallization is extremely laborious; it often involves several scientists entire careers.
b Octylglucopyranoside, a detergent used in membrane protein research
(from the 1997 Aldrich Chemical Catalog)

5. And then... Membrane protein crystallization in cubo
In late 1996, a Swiss group led by E. M. Landau dropped a bombshell on the world of membrane protein crystallography. They had succeeded in crystallizing a bacterial light harvesting protein out of a lipid cubic liquid crystalline matrix.
bR crystals, (3)
The principle is quite simple: if you want to make a membrane protein stable, why not put it in a membrane? But the task of crystallization is more difficult than that. Purity of the protein in the crystal is paramount, and in any case, one must deliver the protein to the artificial membrane somehow. Once the protein is in the membrane, it then must come out and crystallize. These are not trivial matters.
What interests our group is that, though the Landau group has succeeded in extending their technique to a handful of other proteins in a mere few years, no one yet knows how the technique actually works. One idea, pictured at left, depends directly on the chemical precipitants Landau’s group used.
A hypothetical sketch of protein crystals forming from a lipidic cubic phase, (1)

6. Our project
There are two intriguing questions that are raised by the Landau groups experiments. The first is whether the cubic structure they used is important, or whether simply being in a membrane allows the membrane protein to crystallize. Second, and more fundamental, is how the protein actually forms crystals. There are several clues to how this might work, but there are no hard answers.
(1)
It may be that the precipitant is not directly responsible for the change in solubility that leads to crystallization. There is indirect evidence that the crystals form due to structural changes in the surrounding lipid matrix. We are exploring whether changes in lipid crystal phase and lattice parameters lead to protein crystal formation.
Two important lipid phases, (1)
One of the challenges we face is that the complicated detergent-salt system which permits us to solubilize the protein initially can interfere with the structural behavior of the lipids. Indeed, the designer detergents used in membrane protein experiments are designed to have properties quite similar to those of lipids. It is now well known that these detergents do alter the phase behavior of common lipids significantly. However, it may be possible to avoid the use of these detergents entirely. This is a question which remains unanswered.

7. References
1. Caffrey, M, Current Opinion in Structural Biology, 2000, 10:
2. Bowie, JU, Current Opinion in Structual Biology, 2000, 10:
3. Landau, EM and JP Rosenbusch, PNAS, 1996, 93:
4. Pebay-Peyroula, E et al, Biochimica et Biophysica Acta, 2000,
5. Rummel, G et al, Journal of Structural Biology, 1998, 121:82-91
6. Chiu, ML et al, Acta Crystallographica, 2000, D56: