Protein crystallization nucleation

A. M. Kierzek, W. M. Wolf, P. Zielenkiewicz. Simulations of nucleation and early growth stages of protein crystals. Biophys J 75 571, 1997. 

McPherson, A. and Shlichta, P. (1988). Heterogeneous and epitaxial nucleation of protein crystals on mineral surfaces. Science 239, 385-387. 

Haas, C. Drent, J. The Interface between a Protein Crystal and an Aqueous Solution and Its Effects on Nucleation and Crystal Growth. J. Phys. Chem. B 2000, 104, 368-377. 

D Arcy, A., MacSweeny, A., Stihle, M. and Haber, A. (2003). Using natural seeding material to generate nucleation in protein crystallization experiments. Acta Crystallogr. D 59,1343-1346. 

Lorber, B., Jenner, G. and Giege, R. (1996). Effect of high hydrostatic pressure on nucleation and growth of protein crystals. /, Cryst. Growth 158,103-117. 

Saridakis, E. and Chayen, N. E. (2000). Improving protein crystal quality by decoupling nucleation and growth in vapor diffusion. Protein Sci. 9, 755-757. 

The lipidic cubic phase has recently been demonstrated as a new system in which to crystallize membrane proteins [143, 144], and several examples [143, 145, 146] have been reported. The molecular mechanism for such crystallization is not yet clear, but the interfacial water and transport are believed to play an important role in nucleation and crystal growth [146, 147], Using a related model system of reverse micelles, drastic differences in water behavior were observed both experimentally [112, 127, 128, 133-135] and theoretically [117, 148, 149]. In contrast to the ultrafast motions of bulk water that occurs in less than several picoseconds, significantly slower water dynamics were observed in hundreds of picoseconds, which indicates a well-ordered water structure in these confinements. 

Carsten Jacobsen (Novo Nordisk) presented results on protein crystallization in preclarified, concentrated fermentation broths. In particular, the impact of filtration rate on the formation of favorable large diamond versus rod shapes was examined. By adding seed crystals just above the solubility curve, where no nucleation occurred, the authors were able to produce 30% larger crystals as compared to an unseeded crystallization. Although there was minimal recovery and characterization data, this technique may prove very beneficial for dealing with difficult feed streams. While the work presented in this talk was done at the laboratory scale, scale-up experiments will be required to confirm the suitability of this approach for industrial process applications. 

The simplest technique used to grow protein crystals is the batch method in which the protein is mixed with salts or other precipitants to achieve supersaturation (Fig. 2), and the vessel is sealed and set aside until crystals appear. Frequently, the supersaturation point required to induce nucleation is empirically determined by observing the onset of transient turbidity as powdered salt is progressively added to the solution. Crystals of hen egg white lysozyme used for most systematic studies of protein crystallization are grown by batch methods (Blundell and Johnson, 1976). Mouse pancreatic ribonuclease (Perry and Palmer, 1988) and the biotin operon repressor (Brennan et al., 1989) represent recent examples of use of the batch method. 

The objective of most protein crystallization experiments is to obtain a few large crystals. As outlined in Sections IV,C and VI, two of the major obstacles to controlled protein crystal growth are the extreme sensitivity of nucleation rate to supersaturation conditions and the necessity for higher supersaturations to promote nucleation than are needed for growth (Fig. 2). An inherent shortcoming of many crystallization methods is that they depend on similar conditions both to promote nucleation and to support growth. A frequent result is either no crystals or the formation of many small crystals. However, alternative approaches have been developed that attempt to individually optimize nucleation and growth conditions. 

Rosenberger, F. Vekilov, P.G. Muschol, M. Thomas, B.R. Nucleation and crystallization of globular proteins-What we know and what is missing. J. Cryst. Growth 1996, 168 (1 ), 1-27. 

Alexander McPherson and Paul J. Shlichta have suggested using insoluble minerals as heterogeneous nuclei for the crystallization of macromolecules. They obtained excellent protein crystals, which could be cleaved from the mineral nucleus and used for X-ray diffraction studies. The mineral is introduced into a supersaturated solution of the material to be crystallized. As supersaturation increases, nucleation occurs on a specific face of the mineral nucleus, and a crystal begins to grow. The orientation and periodicity of the molecules on the nucleus surface promote an oriented overgrowth that has a similar periodicity. 

McPherson, A., and Shlichta, P. J. Facilitation of the growth of protein crystals by heterogeneous/epitaxial nucleation. J. Crystal Growth 85, 206-214 (1987). 

Tsunekawa S, Ito S, More T, Ishikawa K, Li Z-Q, Kawazoe Y (2000a) Critical size and anomalous lattice expansion in nanocrystalline BaTiOs particles. Phys Rev B 62 3065-3070 Tsunekawa S, Ishikawa K, Li Z-Q, Kawazoe Y, Kasuya A (2000b) Origin of anomalous lattice expansion in oxide nanoparticles. Phys Rev Letters 85 3440-3443 Turkovic A, Ivanda M, Popovic S, Tonejc A, Gotic M, Dubcek P, Music S (1997) Comparative Raman, XRD, HREM and SAXS studies of grain sizes in nanophase Ti02. J Molec Struct 410/411 271-273 ten Wolde PR, Frenkel D (1997) Enhancement of protein crystal nucleation by critical density fluctuations. Science 277 1975-1978... 

A common problem in macromolecular crystallization is inducing crystals to grow that have never previously been observed. The single major obstacle to obtaining any crystals at all is, however, ensuring the formation of stable nuclei of protein crystals. In cases where the immediate problem is growing crystals, attention must be thus directed to the nucleation problem, and any approach that can help promote nucleation should be considered. 

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