The Contribution of Polystyrene Nanospheres towards the Crystallization of Proteins (original) (raw)
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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.
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.
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 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
Enzyme and Microbial Technology, 2002
We demonstrate the feasibility of growing crystals of protein in volumes as small as 1 nanoliter. Advances in the handling of very small volumes (i.e. through inkjet and other technologies) open the way towards fully automated systems. The rationale for these experiments is the desire to develop a system that speeds up the structure determination of proteins by crystallographic techniques, where most of the precious protein sample is wasted for the identification of the ideal crystallisation conditions. An additional potential benefit of crystallisation in very small volumes is the potential improvement of the crystal quality through reduced convection during crystal growth. Furthermore, in such small volumes even very highly supersaturated conditions can be stable for prolonged periods, allowing additional regions of phase-space to be prospected for elusive crystallisation conditions. A massive improvement in the efficiency of protein crystallogenesis will cause a paradigm shift in the biomolecular sciences and will have a major impact in product development in (for example) the pharmaceutical industry.
An Overview of Biological Macromolecule Crystallization
International Journal of Molecular Sciences, 2013
The elucidation of the three dimensional structure of biological macromolecules has provided an important contribution to our current understanding of many basic mechanisms involved in life processes. This enormous impact largely results from the ability of X-ray crystallography to provide accurate structural details at atomic resolution that are a prerequisite for a deeper insight on the way in which bio-macromolecules interact with each other to build up supramolecular nano-machines capable of performing specialized biological functions. With the advent of high-energy synchrotron sources and the development of sophisticated software to solve X-ray and neutron crystal structures of large molecules, the crystallization step has become even more the bottleneck of a successful structure determination. This review introduces the general aspects of protein crystallization, summarizes conventional and innovative crystallization methods and focuses on the new strategies utilized to improve the success rate of experiments and increase crystal diffraction quality.
Crystalline Proteins as an Alternative to Standard Formulations
Chemical Engineering & Technology, 2008
Crystalline proteins may offer superior properties for drug delivery compared to standard protein formulations such as aqueous solutions or amorphous precipitated lyophilisates. In this study, a new approach using biocompatible, hydrophilic, substituted alkylammonium-based ionic liquids (ILs) as additives for the advanced crystallization of two exemplary proteins, lysozyme and lipase, was investigated. Sitting-drop vapor diffusion crystallization experiments revealed that the addition of some of the ILs resulted in less crystal polymorphism and precipitation was consistently avoided, even at larger concentrations of the conventional crystallization agent. The kinetics of lysozyme crystallization were significantly enhanced by a factor of up to 5.5 using ILs with strongly hydrated anions, i.e., formate or glycolate. In contrast, ILs with weakly hydrated anions, i.e., nitrate, led to undesirable spontaneous precipitation. In addition, lipase was crystallized preferentially using an IL with a strongly hydrated anion, i.e. dihydrogenphosphate. Large, sturdy crystals were formed at rates which were enhanced by a factor of up to 4.
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.
Protein crystallization benefits from the rough well surface of a 48-well polystyrene microplate
Journal of Crystal Growth, 2020
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