Protein crystallization nucleation rate

Crystal nucleation rates, expressed as the number of nuclei formed per unit volume per unit time, increase with protein solubility. Higher solubility leads to increased molecular encounters in solution and reduced levels of supersaturation required for spontaneous nucleation. Nucleation rates typically show a high-power dependence on protein supersaturation, and so empirically increase rapidly above a critical value... 

Nucleation of protein crystals typically requires extremely high supersaturation levels. Studies of protein nucleation are limited, with most efforts focused on light scattering as a tool to detect nucleation. Feher and Kam s work set the tone for much of the work that followed (Feher and Kam 1985). They model nucleation in a classical fashion, as a cooperative step-by-step addition of monomers to a cluster. Light scattering is utilized to follow the cluster size distribution as a function of time and solution variables, which yield estimates for the relative forward (cluster growth) and reverse (cluster dissolution) rates of monomer addition. Certainly, the protein crystal nucleation is an area that deserves additional study. 

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. 

Rates of crystal nucleation and growth generally have different dependencies on protein supersaturation, and additionally vary substantially for different protein—precipitant systems. This can lead to a variety of unexpected behaviors in crystallization experiments. The nucleation... 

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. 

As in any crystallization, the production of protein crystals requires bringing the protein into a supersaturated liquid state. The degree of supersaturation determines the rate of nucleation as well as crystal growth rate. Each of these phenomena are... 

FIGURE 10.12 Theoretical and experimental nucleation rate of lysozyme crystals as a function of the Wenzel roughness coefficient (lysozyme dissolved in 0.05 M NaAc buffer at pH 4.5 a precipitant solution prepared by dissolving NaCl in NaAc buffer. Both solutions filtered and mixed 1 1 to give a final NaCl concentration of 2 wt% a final protein concentration of 40 mg/ml). (Adapted with permission from Cutcio et al., J. Phys. Ghent. B, 114,13650-13655. Copyright 2010 American Chemical Society.)... 

Curcio, E., Fontananova, E., Di Rrofio, G. and DrioU, E. 2006. Influence of the structural properties of poly(vinyhdene fluoride) membranes on the heterogeneous nucleation rate of protein crystals. 110 12438-12445. 

Changes in a single experimental parameter can simultaneously influence several aspects of a crystallization experiment. For example, temperature changes affect protein solubility, rates of nucleation and growth, and equilibration of the experimental apparatus. The interaction of parameters makes it difficult to design experiments to isolate individual effects and likewise complicates the interpretation of experimental results. 

Once conditions for nucleation and growth have been identified and the investigation of variables more or less complete, the concentration of the protein should be gradually reduced in increments to moderate the growth of the crystals. As a rule, the largest and most perfect crystals result when the rate of incorporation of molecules is slow and orderly. Reduction of macromolecule concentration is an effective means for controlling this. 

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