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2] PROTEIN CRYSTALLIZATION METHODS 13 [2] Overview of Protein Crystallization Methods
In~oducUon Crystallographic structure determination begins with growth of a suitable crystal. With the availability of powerful X-ray sources, rapid data collection instruments, and faster computers, crystallization has increasingly become the rate-limiting step in macromolecular structure determinations. In this chapter, some of the physical principles that govern crystal growth are presented to assist the crystallographer in designing crystallization experiments and interpreting their results. The appearance of a macroscopic protein crystal containing roughly 1015 molecules begins with association of protein aggregates whose intermo-lecular contacts resemble those found in the final crystal. 1' 2 These prenuclear aggregates eventually reach the critical nuclear size. Given stable nuclei, growth proceeds via addition of molecules to the crystalline lattice. Both crystal nucleation and growth occur in supersaturated solutions where the concentration of protein exceeds its equilibrium solubility value. The region of solution parameter space suitable for crystallization is generally represented on the phase diagram by the solubility curve (Fig. 1). Supersaturation is a function of the concentration of the macromolecule and parameters that affect its solubility. It is achieved at high macromolecu-lar concentrations, and at increasing values of solution parameters that decrease macromolecular solubility. Many factors can influence protein solubility. Inclusion of additives such as alcohols, hydrophilic polymers, and detergents can decrease protein solubility. While these are commonly referred to as precipitants, they are solubility-influencing agents, with protein precipitation being only one possible outcome of their addition to the solution. Protein solubility as a function of salt is usually an asymmetric bell-shaped curve with decreased solubility at both high and low salt concentrations. Consequently, strategies involving both inclusion and exclusion of salt can induce protein crystallization. Solution parameters such as pH or temperature can also dramatically influence macromolecular solubility.
Important Factors Influencing Protein Crystallization
The solution of crystallization problem was introduced around twenty years ago, with the introduction of crystallization screening methods. Here reported some of the factors which affect protein crystallization, solubility, Concentration of precipitant, concentration of macromolecule, ionic strength, pH, temperature, and organism source of macromolecules, reducing or oxidizing environment, additives, ligands, presence of substrates, inhibitors, coenzymes, metal ions and rate of equilibration. The aim of this paper to give very helpful advice for crystallization.
Crystal Growth & Design, 2008
This work combined water equilibration fundamentals of vapor diffusion crystallization techniques with protein solubility data in order to obtain the variation of protein supersaturation throughout the protein crystallization assays. Once the supersaturation build up profiles (SBUPs) are known, the wide screening space of crystallization conditions is reduced to the key variable for crystal formation and growth, which is supersaturation and its variation with time. Our previous water equilibration model was expanded to include the case of drop evaporation at constant contact area during the hanging drop method. Crystallization experiments of lysozyme were performed under different experimental conditions and the results were interpreted according to the respective SBPUs. In particular, the number and size of the crystals were evaluated at the moment of the SBUP that corresponded to their formation. Following this methodology, two nucleation behaviors were identified depending on the supersaturation levels at which crystal formation occurs. These behaviors, which are believed to be closely linked with the diffracting properties of the crystals, are dictated not only by classic thermodynamic and kinetic factors affecting crystallization and water equilibration, but also by phenomena related to the drop preparation procedures.
Introduction to protein crystallization
Methods, 2004
Biological macromolecules can be crystallized by a variety of techniques, and using a wide range of reagents which produce supersaturated mother liquors. These may, in turn, be applied under diVerent physical conditions such as temperature. The fundamental approaches to devising successful crystallization conditions and the factors that inXuence them are summarized here. For the novice, it is hoped that this brief review might serve as a useful introduction and a stepping-stone to a successful X-ray strucutre determination. In addition, it may provide a framework in which to place the articles that follow.
Journal of Crystal Growth, 2003
The combined effect of both pressure and pH on the crystallization in agarose gel of thaumatin (0.1-230 MPa and pH 5.7-8.0) and the lysozymes from turkey (0.1-100 MPa and pH 4.0-6.0) and hen egg-white (0.1-75 MPa and pH 4.0-6.0) was investigated. For each condition, crystal morphology was examined and four crystal growth parameters determined (crystal number as a measure of nucleation, solubility, supersaturation and pressure-induced volume changes of crystallization). Comparison of data reveals a different crystallization behavior of thaumatin and the two lysozymes. First, thaumatin is more pressure tolerant in agarose gel than the lysozymes, with crystals growing up to 230 MPa for the former and only up to 75/100 MPa for the latter. Second, while an increase in pressure and pH increases nucleation of thaumatin crystals and decreases their solubility, one observes an inverse effect with the two lysozymes. Here, an increase in pressure but a decrease in pH increases nucleation and solubility. Third, pH influences the pressure-induced volume changes of crystallization, the strongest pH-dependence being found for turkey lysozyme. However, the volume changes are negative for thaumatin and positive for both lysozymes. These volume changes are correlated with solubility changes and are explained by pH-and pressure-induced alterations of the conformation of the three proteins, including their solvation shells. r
Enzyme and Microbial Technology, 2002
Proteins and enzymes for commercial use are often dried in order to extend their shelf lives. This study examined the effects of disaccharides (sucrose and trehalose), polymers (dextran and maltodextrin) and disaccharide-polymer mixtures on the stability of subtilisin, a common industrial laundry detergent enzyme, during drying and during subsequent storage in dried solids containing perborate, a laundry detergent additive that hydrolyzes to form hydrogen peroxide in the presence of water. Subtilisin is susceptible to oxidation by hydrogen peroxide at a partially buried methionine residue, which results in loss of catalytic activity. Two drying methods were compared: spray coating and lyophilization. During both drying methods, the presence of a disaccharide, which can hydrogen bond to subtilisin in the place of lost water, was necessary to inhibit dehydration-induced unfolding of subtilisin. Dehydration-induced unfolding was assessed by second-derivative IR spectroscopy. However, even in the samples in which the protein's secondary structure was perturbed, the protein refolded to the native conformation and exhibited full activity upon rehydration. None of the additives protected Met222 from oxidation during storage of the initially dry protein formulations at 37 • C and 20 or 75% relative humidity under oxidizing conditions. In contrast, the two additional methionine residues in the enzyme, Met119 and Met175, which are deeply buried in the interior of the protein, remained unmodified during storage of the dried formulations and became oxidized only if the enzyme was completely unfolded in aqueous solution.
Activity and Stability of Native and Modified Subtilisins in Various Media
Biochemistry (Moscow), 2000
The activity and stability of native subtilisin 72, its complex with poly(acrylic acid), and subtilisin covalently attached to poly(vinyl alcohol) cryogel were studied in aqueous and organic media by hydrolysis of specific chromogenic pep tide substrates. Kinetic parameters of the hydrolysis of Glp Ala Ala Leu pNA by native subtilisin and its complex with poly(acrylic acid) were determined. Based on the comparative study of stability of native and modified subtilisins in media of various compositions, it was established that covalent immobilization of subtilisin on poly(vinyl alcohol) cryogel is the most effective approach to improve enzyme stability in water as well as in mixtures with low water content.
Development of an alternative approach to protein crystallization
Journal of Structural and Functional Genomics, 2007
We are developing an alternate strategy for the crystallization of macromolecules that does not, like current methods, depend on the optimization of traditional variables such as pH and precipitant concentration, but is based on the hypothesis that many conventional small molecules might establish stabilizing, intermolecular, non covalent crosslinks in crystals, and thereby promote lattice formation. To test the hypothesis, we carried out preliminary experiments encompassing 18,240 crystallization trials using 81 different proteins, and 200 chemical compounds. Statistical analysis of the results demonstrated the validity of the idea. In addition, we conducted X-ray diffraction analyses of some of the crystals grown in the experiments. These clearly showed incorporation of conventional molecules into the protein crystal lattices, and further validated the underlying hypothesis. We are currently extending the investigations to include a broader and more diverse set of proteins, an expanded search of conventional and biologically active small molecules, and a wider range of precipitants. The strategy proposed here is essentially orthogonal to current approaches and has an objective of doubling the success rate of today.
Journal of Applied Crystallography, 2005
High-throughput screening of a wide range of different conditions is typically required to obtain X-ray quality crystals of proteins for structure-function studies. The outcomes of individual experiments, i.e. the formation of gels, precipitates, microcrystals, or crystals, guide the search for and optimization of conditions resulting in X-ray diffraction quality crystals. Unfortunately, the protein will remain soluble in a large fraction of the experiments. In this paper, an evaporation-based crystallization platform is reported in which droplets containing protein and precipitant are gradually concentrated through evaporation of solvent until the solvent is completely evaporated. A phase transition is thus ensured for each individual crystallization compartment; hence the number of experiments and the amount of precious protein needed to identify suitable crystallization conditions is reduced. The evaporation-based method also allows for rapid screening of different rates of supersaturation, a parameter known to be important for optimization of crystal growth and quality. The successful implementation of this evaporation-based crystallization platform for identification and especially optimization of crystallization conditions is demonstrated using the model proteins of lysozyme and thaumatin. research papers J. Appl. Cryst. (2005). 38, 988-995 Sameer Talreja et al. Protein crystallization conditions 995 electronic reprint