Development of an alternative approach to protein crystallization

Abstract

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.

Crystal cookery – using high-throughput technologies and the grocery store as a teaching tool

“…Where would we go for the next step? We have postulated that some of the PSBs may act in the same proteinspecific manner as the real Silver Bullets (Larson et al, 2007a;McPherson & Cudney, 2006;McPherson et al, 2007). We have circumstantial evidence but no proof that this is the case.…”

“…We saw that the former was true in the DSF experiments. However, our implicit assumption that some of these grocery store products could act in a similar manner to the commercially available Silver Bullets (Larson et al, 2007a;McPherson & Cudney, 2006;McPherson et al, 2007) was not rigorously tested with crystallographic analysis of the structures. While we certainly observed crystallization hits from the PSB experiments, we do not know if this was through specific small-molecule interactions with the protein, as associated with true silver bullets, or a more general chemical effect caused by ingredients that altered the solubility of the protein in a less specific manner, e.g.…”

“…While supersaturation of crystallization experiments is most commonly achieved using chemical precipitants, variables such as pH (McPherson, 1995), temperature (Astier & Veesler, 2008) and even pressure (Visuri et al, 1990) have been used to promote crystallization. An alternative crystallization strategy, Silver Bullets (Hampton Research, Aliso Viejo, California, USA), uses libraries of small molecules (inorganic and organic salts, organic acids, biological ligands, amino acids, and peptides) to stabilize and promote lattice contacts in order to crystallize biological macromolecules using a very limited set of supersaturating conditions (Larson et al, 2007a;McPherson & Cudney, 2006;McPherson et al, 2007). The macromolecules select specific chemically relevant small molecules from these multiple component solutions.…”

An Analysis on Commercial Screening Kits and Chemical Components in Biomacromolecular Crystallization Screening

“…For example, a certain concentration of acetone can increase the crystallization efficiency of insulin fivefold higher than with control treatment . Except for detergents, small molecular additives such as coenzymes, substrate analogues, and inhibitors can bind with biomacromolecules with the purpose of forming homogeneous and stable structures, leading to desirable crystallization . Other molecules such as sugars, proline, glycerol, and betaine may impact biomacromolecular crystallization by altering the interaction between the surface of sample and water to reform the inherent structure of macromolecules …”

A protein crystallisation screening kit designed using polyethylene glycol as major precipitant

“…20 Tacsimate exhibits similar properties to those of sodium malonate and potassium sodium tartrate tetrahydrate and showed powerful effects on crystallisation screening experiments. 21,22 These organic acid salts were also chosen as additives.…”