Crystallization of lysozyme with (R)-, (S)- and (RS)-2-methyl-2,4-pentanediol

Abstract

Chiral control of crystallization has ample precedent in the small-molecule world, but relatively little is known about the role of chirality in protein crystallization. In this study, lysozyme was crystallized in the presence of the chiral additive 2-methyl-2,4-pentanediol (MPD) separately using the R and S enantiomers as well as with a racemic RS mixture. Crystals grown with (R)-MPD had the most order and produced the highest resolution protein structures. This result is consistent with the observation that in the crystals grown with (R)-MPD and (RS)-MPD the crystal contacts are made by (R)-MPD, demonstrating that there is preferential interaction between lysozyme and this enantiomer. These findings suggest that chiral interactions are important in protein crystallization.

Keywords: MPD; chirality; crystallization additives; high-resolution protein structures; precipitants.

Figure 1

Schematic representation of (R)- and (S)-MPD. The C atoms are numbered according to the convention used in this work and the torsion angles (ψ₁, ψ₂) are represented by red arrows. CM is the unlabelled methyl C atom attached to C2 by a dashed line; the O atoms attached to C2 and C4 are O2 and O4, respectively, and the chiral center is at C4.

Figure 2

The conformers of MPD studied by quantum-chemical calculations. Each conformer is denoted by a two-symbol code (number and letter) that approximately represents the torsion angles (ψ₁, ψ₂) of the initial conformation used in the calculation. The numbers 1, 2 and 3 correspond to ψ₁ = 180, 300 and 60°, respectively; the letters a, b and c correspond to ψ₂ = 180, 60 and 300°, respectively. [For example, 1a is the (180°, 180°) conformer shown in Fig. 1 ▶.] To interconvert two adjacent structures, a rotation is performed about the carbon–carbon bond given in square brackets. For clarity, only C2, C3 and C4 are numbered; C1 is shown in red, while CM and C5 (which is adjacent to C4) are shown in black.

Figure 3

The conformations of MPD from MD simulations in vacuo. (a) (R)-MPD at 300 K; (b) (R)-MPD at 370 K; (c) (S)-MPD at 300 K; (d) (S)-MPD at 370 K. In (a) the dashed lines mark the nine bins used in the free-energy calculations. The red circles are the nine locally stable conformers obtained from quantum-chemical calculations; the corresponding label for each conformer is shown in bold. The results of the simulated-annealing experiments lie within the green squares.

Figure 4

The conformations of MPD from the PDB. The two enantiomers are shown in different colors: (R)-MPD in red and (S)-MPD in blue. The dashed lines mark the nine bins used in the free-energy calculations (cf. Fig. 3 ▶ a).

Figure 5

Overall structures of lysozyme with the enantiomers of MPD. A ribbon diagram of the C^α backbone is shown for crystals grown with (R)-MPD (blue) and (S)-MPD (green). The MPD molecules associated with each structure are shown in the same color as the protein backbone.

Figure 6

The crystal contact site near Phe34. Two symmetry-related protein molecules (shown with green and purple C atoms) interact through hydrogen bonds (dashed orange lines). (a)–(d) depict the results from the current work with different additives: (a) (R)-MPD; (b) (S)-MPD; (c) (RS)-MPD; (d) no MPD added. The red sphere in (d) is the O atom of a water molecule. For comparison, the results of (e) Weiss et al. (2000 ▶) and (f) Michaux et al. (2008 ▶), both of which used (RS)-MPD, are also presented. The 2F _o − F _c density is shown as a gray mesh contoured at 1.5σ.

Figure 7

The interaction site near Trp63. (a)–(c) depict the results from the current work with different additives: (a) (R)-MPD; (b) (S)-MPD; (c) (RS)-MPD. The red spheres in (b) are the O atoms of water molecules. For comparison, the results of Michaux et al. (2008 ▶), which used (RS)-MPD, are also presented (d). The 2F _o − F _c density is shown as a gray mesh contoured at 1.5σ, except for the (S)-MPD molecule in (d), which is shown at 1.0σ. This is to highlight that C4 (denoted by an arrow) protrudes from the density.

Figure 8

The interaction site near Trp123. (S)-MPD interacts with the protein through hydrogen bonds (dashed orange lines). The red sphere is the O atom of a water molecule. The 2F _o − F _c density is shown as a gray mesh contoured at 1.5σ. For clarity, the intramolecular hydrogen bond between O2 and O4 of (S)-MPD is not shown.