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Membrane Protein Crystallization From
Cubic Lipid Matrices
Marcus D. Collins and Sol M. Gruner,
Cornell University LASSP
Membrane proteins at a glance.
What makes membrane proteins hard to crystallize?
The x-ray determined structure of the light harvesting protein bacteriorhodopsin, from
Halobacterium halobium, (5)
What has succeeded?
The open questions and our project.
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
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.
Various membrane proteins shown from views parallel (above)
and perpendicular to the membrane, (6)
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.
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.
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.
b Octylglucopyranoside, a detergent used in membrane protein research
(from the 1997 Aldrich Chemical Catalog)
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.
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)
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
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
1. Caffrey, M, Current Opinion in Structural Biology, 2000, 10:486-497
2. Bowie, JU, Current Opinion in Structual Biology, 2000, 10:435-437
3. Landau, EM and JP Rosenbusch, PNAS, 1996, 93:14532-14535
4. Pebay-Peyroula, E et al, Biochimica et Biophysica Acta, 2000, 119-132
5. Rummel, G et al, Journal of Structural Biology, 1998, 121:82-91
6. Chiu, ML et al, Acta Crystallographica, 2000, D56:781-784