BioPHYSICAL chemistry

protein. Thus, in most cases, the protein precipitates and crystals are obtained

only by surveying many different solvent conditions in the hope that one

favors the slow and ordered growth needed for crystallization.

The preferred approach for producing protein crystals is to use vapor

diffusion, in which the process is driven by the removal of water molecules

from the protein solution. Two solutions are placed in a closed container:

a reservoir and a protein solution. The reservoir contains the precipitat-

ing agent, usually a salt or polyethylene glycol, and has a relatively large

volume (typically 1 mL). The second solution has the protein, buffer, and

a smaller concentration of the precipitating agent, and a much smaller

volume (about 1μL). Because both solutions are in the same closed con-

tainer, each solution will equilibrate through the vapor exchange between

solutions. With time the liquid from the small drop will change into vapor

and recondense with the large solution. This process will continue until

equilibrium is achieved. At equilibrium, the chemical potentials associated

with the precipitant are equal, hence the precipitant concentrations are

the same. Thus, the protein drop has been concentrated by a factor given

by the ratio of the initial precipitant concentrations of the two solutions.

For example, if initially the salt concentrations are 0.5 and 1.0 M for the

protein drop and reservoir, respectively, the volume of the protein drop

will be reduced by half and the concentrations of the protein and other

components will be doubled (so that the final salt concentration in the

protein drop will be 1.0 M, matching the reservoir concentration).

Why does this lead to crystallization? Vapor diffusion causes a slow

decrease in the amount of water in the small drop and hence a slow increase

in the concentrations of the protein and precipitating agent (as well as the

buffer). The dependence of the state of the protein, either in solution or

in a crystal, on the salt and protein concentration can be mapped out with

a phase diagram (Figure 4.18). On these diagrams, the lines represent

90 PARTI THERMODYNAMICS AND KINETICS

Initial solution Final solution Unsaturation

Supersaturation

[Macromolecule]

Solubility

Figure 4.18The crystallization process using vapor diffusion can be described in terms of a phase
diagram.