Protein crystallization using room temperature ionic liquids

The ionic liquids (ILs) 1-butyl-3-methylimidizolium chloride ([C(4)mim]Cl), 1-butyl-3-methylimidizolium 2(2-methoxyethoxy)ethylsulfate ([C(4)mim][MDEGSO4]), and 1-butyl-1-methylpyrollidinium dihydrogenphosphate ([p1,4][DHP]) were tested for their effects on the crystallization of the proteins canavalin, beta-lactoglobulin B, xylanase, and glucose isomerase, using a standard high throughput screen. The crystallization experiments were set up with the ILs added to the protein solutions at 0.2 and 0.4 M final concentrations. Crystallization droplets were set up at three protein/precipitant ratios (1:1, 2:1, and 4:1), which served to progressively dilute the effects of the screen components while increasing the equilibrium protein and IL concentrations. Crystals were obtained for all four proteins at a number of conditions where they were not obtained from IL-free control experiments. Over half of the protein-IL combinations tested had more successful outcomes than negative outcomes, where the IL-free crystallization was better than the corresponding IL-containing outcome, relative to the control. One of the most common causes of a negative outcome was solubilization of the protein by the IL, resulting in a clear drop. In one instance, we were able to use the IL-induced solubilizing to obtain beta-lactoglobulin B crystals from conditions that gave precipitated protein in the absence of IL. The results suggest that it may be feasible to develop ILs specifically for the task of macromolecule crystallization.