Two-Dimensional Crystallization of Biological Macromolecules - Molecules: Nucleation, Aggregation and Crystallization - page 98

stage it may be possible to draw important conclusions with regard to unit
cell size, symmetry and possible stacking.
How to Make 2D Crystals of Proteins
For the purpose of discussing 2D crystallization of proteins it is appropri-
ate to subdivide these biological macromolecules into two categories: sol-
uble proteins and membrane proteins. The reason for this is that two
completely different strategies are dominating for handling the two sub-
types. However, the subdivision is not static. Although all soluble proteins
can be dissolved in a water solution, some of them, like pore-forming bac-
terial toxins, may be able to change conformation for accommodation in
a lipophilic environment. Integral membrane proteins of type I and type II
with only one membrane spanning segment may be transferred into a sol-
uble protein by cleavage of the membrane anchor. Since 2D crystalliza-
tion has been most frequently applied to membrane proteins, those will be
treated first and with most detail in the following.
Membrane proteins
2D crystallization of membrane proteins can proceed in two different
ways depending on the specimen. Most frequently the starting material is
a purified membrane protein dissolved in a detergent-containing buffer.
The other category is proteins which are embedded in a natural membrane
and suspended in a buffer. The dominating purified specimens will be
treated first.
The basic idea behind forming 2D crystals from membrane proteins
in detergent solutions is to add phospholipids solubilized in detergent and
reduce the detergent concentration of the mixture (for a recent review, see
Schmidt-Krey (2007)). Under proper conditions phospholipid bilayers
will form with inserted protein molecules which arrange themselves into
a crystalline sheet (Fig. 1). The reduction of the detergent concentration is
normally done either by dialysis or adsorption onto biobeads. Dialysis can
be performed with different types of containers such as dialysis tubes,