A novel strategy for the crystallization of proteins: X-ray diffraction validation

Abstract

Recently, the hypothesis was advanced that protein crystallization could be driven by the inclusion of small molecules rich in hydrogen‐bonding, hydrophobic and electrostatic bonding possibilities. Conventional organic and biologically active molecules would promote lattice formation by their mediation of intermolecular interactions in crystals. The results of an extensive series of crystallization experiments strongly supported the idea. Here, difference Fourier X‐ray diffraction analyses of nine crystals grown in the experiments are presented, which convincingly demonstrate the validity of the hypothesis and illustrate some of the ways in which small molecules can participate in lattice interactions.

Protein Recognition by Functionalized Sulfonatocalix[4]arenes

“…As a charge‐dense, anionic dimer Br.sclx may serve as “filler” in the crystal packing of cationic proteins. This molecular glue functionality is supported by McPherson's demonstration that the incorporation of small molecules into the crystal lattice can promote protein crystal growth . In the case of Ph.sclx, the crystal packing involved two ligands in close proximity such that the phenyl substituents were in van der Waals contact (Figure B) and packed against a hydrophobic patch comprising Ala3, Ala7, Tyr97, the C γ of T98, and Ala101.…”

An Overview of Biological Macromolecule Crystallization

“…Recent studies have shown that the application of biocompatible water-soluble ionic liquids, organic salts and salts with melting points at or below room temperature as crystallization additives provides very interesting results [39,40]. Small organic molecules represent another class of very useful additives, which increase long range electrostatic interactions by lowering the dielectric constant, affect solvent structure, and could modify the hydrophobic effect [41–45]. Crystallization of a certain protein often requires the presence in the crystallizing solution of natural ligands, which assure the conformational homogeneity of the sample.…”

Development of an alternative approach to protein crystallization

“…Only by this technique could detailed interactions within lattices be directly visualized, and the original idea rigorously evaluated. In a subsequent paper [2] we described analyses of nine crystalline proteins grown in the original experiments, each obtained in the presence of a ''cocktail'' of low molecular weight compounds. Another, independent analysis was carried out on bovine trypsin crystals grown in our experiments in the presence of banzamidine and protamine [6].…”

“…Subsequent to the screening and X-ray diffraction experiments we have already described [2,4] we have now conducted two additional experiments. Details of those will be presented elsewhere, but a few observations are appropriate here.…”

“…The fundamental hypothesis driving this research is that conventional molecules having unique features may (1) be bound by biological macromolecules, which may then be stabilized or induced to assume a more favorable conformation for crystallization, (2) that the small molecules may alter the interactions between macromolecules and their solvent and induce ordered association, or (3) that the small molecules may form reversible, cross links in a lattice through hydrogen bonding, electrostatic, and possibly hydrophobic interactions, and thereby promote formation of a crystal. There is ample evidence in the literature, and in our preliminary results, to support this hypothesis and suggest that future research may be profitable.…”