Protein crystallization phase diagrams

The crystallization process can be illustrated by a phase diagram that shows which state (liquid, crystalline, or amorphous solid [precipitate]) is stable under a variety of crystallization parameters. It provides a means of quantifying the influence of the parameters such as the concentrations of protein, precipitant(s), additive(s), pH, and temperature on the production of crystals. Hence phase diagrams form the basis for the design of crystal growth conditions (McPherson, 1999 Ducruix and Giege, 1992 Ducruix and Giege, 1999 Chayen et ah, 1996 and references therein). 

Ataka, M. (1993). Protein crystal growth An approach based on phase diagram determination. Phase Transitions 45, 205-219. 

Saridakis, E. and Chayen, N. E. (2003). Systematic improvement of protein crystals by determining the supersolubility curves of phase diagrams. Biophys. 84, 1218-1222. 

Figure 4 Phase diagram for a protein solution. In the undersaturation (soluble) zone crystals do not grow but dissolve the first line marks the saturation limit. Above that, the solution is supersaturated and metastable with respect to the crystals existing crystals will grow, but no spontaneous nucleation occurs. In the nucleation zone, new crystals form on their own, and in the precipitation zone nonspecific aggregation dominates.

Figure 6 Movement on the phase diagram in a vapor diffusion experiment. The simultaneous increase of both precipitant and protein concentrations corresponds to a diagonal movement in the phase diagram. Once crystal nucleation occurs, the growing crystals consume the protein in the solution, until the solution is no longer supersaturated.

The re-entrant or Tammann loop-shape phase diagram as observed in proteins is also found in other systems and has been connected to exothermic disordering [88]. In this particular case, nematic - smectic A transitions in liquid crystals and the phase behaviour of a crystalline polymer, poly(4-methyl-pentene-l), the phase behaviour can be understood by... 

Figure 12.16 The generic protein phase diagram as presented by Muschol and Rosenberger (1997). Zone I depicts the region of supersaturation where well formed lysozyme crystals form. Zone II is a region where lysozyme undergoes a rapid liquid-liquid phase separation, with the resulting concentrated lysozyme phase quickly tranforming to the more stable crystalline form. Crystals formed in Zone II are of poor quality. Zone III depicts a region characterized by gel formation, unsuitable for crystal growth.

The process can be described by reference to the phase diagram (Figure 5.18). At low concentrations of protein and salt (or other precipitant) the protein is below the solubility limit and remains in solution. At high concentrations, however, the solution becomes thermodynamically unstable (supersaturated) and, once nucleation takes place, the protein will precipitate out of solution until the concentration in the liquid phase falls back to the solubility limit. If this happens too quickly, then the precipitate tends to be amorphous or just composed of very tiny crystals. 

It was shown that this remarkable phase behaviour could be understood on the basis of the sensitivity to the form of the pair potential of the phase diagram of small attractive colloidal particles [268-270]. Moreover, it was soon realized that successful protein crystallization depends on the location (protein concentration and temperature) in the phase diagram [271-275]. Control of protein crystal nucleation around the metastable liquid-liquid phase boundary appears key to the development of systematic crystallization strategies (for a concise review see [276]). This phase boundary can be manipulated by depletion interactions through the addition of non-adsorbing polymers such as polyethylene glycol. 

Fig. 3.20 An example of a phase diagram in which the mole fraction of a precipitant, a substance that causes precipitation, is plotted against the mole fraction of a protein. The data help biochemists find conditions under which a protein crystallizes.

---