Precipitation diagram and optimization of crystallization conditions at low ionic strength for deglycosylated dye-decolorizing peroxidase from a basidiomycete

Abstract

The growth of suitably sized protein crystals is essential for protein structure determination by X-ray crystallography. In general, crystals are grown using a trial-and-error method. However, these methods have been modified with the advent of microlitre dispensing-robot technology and of protocols that rapidly screen for crystal nucleation conditions. The use of one such automatic dispenser for mixing protein drops (1.3-2.0 microl in volume) of known concentration and pH with precipitating solutions (ejecting 2.0 microl droplets) containing salt is described here. The results of the experiments are relevant to a crystallization approach based on a two-step procedure: screening for the crystal nucleation step employing robotics followed by optimization of the crystallization conditions using incomplete factorial experimental design. Large crystals have successfully been obtained using quantities as small as 3.52 mg protein.

Figure 1

Precipitation diagrams of deglycosylated DyP at pH 6.0 and 277 K. The precipitation curve, plotted using a full line, was defined by the first signs of light precipitation formation (squares) and then by crystal formation (circles), excluding the rust-coloured clear-drop zones (triangles). Error bars of the x and y axes were estimated from the dispensing accuracy, with standard errors in the PEG 8000 and protein concentrations of 0.18 and 0.07, respectively. The grey-framed rectangle indicates the concentration intervals (filled circles) chosen for optimization with an incomplete factorial design as the nucleation (and growth) zone. The largest single crystal obtained is denoted by a filled diamond. The boundary line between the crystal and clear-drop zones of DyP with PEG 8000 indicates the nucleation concentration curve and is plotted using a dashed line. The middle of the three lines, plotted using a dotted and dashed line, is defined as the boundary between crystal and spherulite formation.

Figure 2

(a) Spherulites on the surface of the drop, which were observed before crystal formation, under a microscope at full-range magnification of 110×. (b) DyP crystal obtained using the sitting-drop vapour-diffusion method. The rust-coloured crystal is in the form of a hexagonal prism and belongs to a monoclinic system.

Figure 3

Response-surface plot of the measured crystal quality versus protein and precipitate concentrations. The average 〈I〉/σ〈I〉 is dependent on DyP and PEG 8000 concentrations. The surface plot was shown in the variable range between −1 [7.48 mg ml⁻¹ DyP, 23.4%(w/v) PEG 8000] and +1 [9.50 mg ml⁻¹ DyP, 27.0%(w/v) PEG 8000]. The maximum of 〈I〉/σ〈I〉 is of 6.8 and represents the optimum point on the surface, indicated by X. The figure was prepared using GNUPLOT (http://www.gnuplot.info).