BioPHYSICAL chemistry

boundaries between a phase where the protein is in solution to where the
protein has become either a precipitant or crystal. With vapor diffusion,
the concentration of both the protein and salt increase simultaneously,
as shown by the diagonal line. The dark line represents the solubility
dependence, so when this line is crossed the protein concentration is above
the solubility, corresponding to a supersaturated solution. Since the super-
saturated solution is not stable, the protein will leave the solution as either
a precipitate or crystals. Phase diagrams such as these are sometimes used
to design better conditions for crystallization.
Crystallization can be enhanced when the crystallization trial is poised
such that the presence of a critical nucleus is favored. One classical approach
is seeding, in which a microscopic protein crystal is inserted into the
crystallization solution. Since the nucleus is already formed, energetically,
the growth of the crystal is favorable. Although this approach can be
highly effective, often the precise conditions needed for the crystal growth
are highly specific and time-consuming to identify. The best seed is to
make use of small protein crystals that one hopes will turn into much
larger crystals. An alternative is to make use of other seed material that
has the proper surfaces, but this has been successful only for small
molecules. More generic approaches have been proposed, such as the
use of porous templates (Page & Sear 2006; Figure 4.19). In this case,
the base material is a porous surface that has a distribution of sizes on the
microscopic level. Among the large number of pores, there will be one
with a size that matches the length needed to accommodate a critical
nucleus. Crystallization starts at one edge of the pore, and protein mole-
cules will attach to the nucleus until the pore is filled. Once the crystal
has grown and filled the pore then the crystallization continues and a
large crystal can be formed. Ideally, the development of tools such as porous
templates or microfluidic chambers (Hansen et al. 2004; Page & Sear 2006)
should help overcome the major bottleneck in the determination of pro-
tein structures.

CHAPTER 4 PHASE DIAGRAMS AND MIXTURES 91

Critical

Molecules nucleus

Pore walls

Critical

nucleus

Figure 4.19Crystal nucleation occurs in the pore of a surface and serves as a seed for growing a
large crystal. Modified from Page and Sear (2006).