Comparative analysis of amino acids and amino-acid derivatives in protein crystallization

Abstract

Optimal conditions for protein crystallization are difficult to determine because proteins tend to aggregate in saturated solutions. This study comprehensively evaluates amino acids and amino-acid derivatives as additives for crystallization. This fourth component of the solution increases the probability of crystallization of hen egg-white lysozyme in various precipitants owing to a decrease in aggregation. These results suggest that the addition of certain types of amino acids and amino-acid derivatives, such as Arg, Lys and esterified and amidated amino acids, is a simple method of improving the success rate of protein crystallization.

Figure 1

Crystallization of 100 mg ml⁻¹ (open bars) or 150 mg ml⁻¹ (shaded bars) HEWL using ammonium sulfate as a precipitant in the presence of 0.25 M amino acid at pH 4.5.

Figure 2

Success rate of crystallization of 25 mg ml⁻¹ (squares) or 50 mg ml⁻¹ (triangles) HEWL in the presence of 0.1 M amino acids at pH 4.5 with 50 unique combinations of sparse-matrix reagents using Crystal Screen I. A reference line across the graph indicates the success rate of the control experiments.

Figure 3

Representative images of HEWL crystals in the presence or absence of amino acids as additives. (a) The crystallization condition was 0.2 M ammonium sulfate, 30% PEG 8000 pH 4.5 with 0.1 M additives. A control crystallization was performed without additives. (b) The crystallization condition was 2.0 M ammonium sulfate, 2% PEG 400, 0.1 M Na HEPES pH 7.5 and 0.1 M Lys.

Figure 4

Concentration ranges of sodium chloride as a precipitant in the presence of 0.25 M additives at pH 6.5 in which crystals were observed using 50 mg ml⁻¹ HEWL after 2 d.

Figure 5

Aggregation of lysozyme under various sodium chloride concentrations in the presence or absence of amino acids and amino-acid derivatives at pH 6.5 and 293 K after 2 d. (a) Turbidity at 600 nm. (b) Supernatant concentration of lysozyme. No additive, open squares; Gly, open triangles; Asp, open circles; Glu, open diamonds; Lys, full squares; Arg, full triangles; GlyEE, full circles; GlyAd, full diamonds.

Figure 6

Comparison of the relationship between the hydrophobicity of each amino acid (Fauchere & Pliska, 1983 ▶) and the success rate of HEWL crystallization. Arg, open square; Lys, open triangle; Asp, open circle; Glu, open diamond; Ser, line; Gly, full square; Thr, full triangle; Ala, full circle; Pro, full diamond; Val, plus symbol.