Lysozyme crystals dyed with bromophenol blue: where has the dye gone?

Abstract

Protein crystals can easily be coloured by adding dyes to their mother liquor, but most structures of these protein–dye complexes remain unsolved. Here, structures of lysozyme in complex with bromophenol blue obtained by soaking orthorhombic and tetragonal crystals in a saturated solution of the dye at different pH values from 5.0 to 7.5 are reported. Two different binding sites can be found in the lysozyme–bromophenol blue crystals: binding site I is located near the amino- and carboxyl-termini, while binding site II is located adjacent to helices α1 (residues 4–15) and α3 (residues 88–100). In the orthorhombic crystals soaked at pH 7.0, binding of the dye takes place in both sites without significant changes in the unit cell. However, soaking tetragonal crystals with bromophenol blue results in two different complexes. Crystals soaked at pH 5.5 (HEWL-T1) show a single dye molecule bound to site II, and the crystals belong to space group P43212 without significant changes in the unit cell (a = b = 78.50, c = 37.34 Å). On the other hand, crystals soaked at pH 6.5 in the presence of imidazole (HEWL-T2) show up to eight molecules of the dye bound to site II, and display changes in space group (P212121) and unit cell (a = 38.00, b = 76.65, c = 84.86 Å). In all of the structures, the dye molecules are placed at the surface of the protein near to positively charged residues accessible through the main solvent channels of the crystal. Differences in the arrangement of the dye molecules at the surface of the protein suggest that the binding is not specific and is mainly driven by electrostatic interactions.

LifeSoaks: a tool for analyzing solvent channels in protein crystals and obstacles for soaking experiments

“…The crystal bottleneck radii are considerably smaller than the minimum projection radius of the dye (5.4, 4.8 and 3.8 A ˚, respectively, versus 5.8 A ˚). The bottleneck radii in front of the binding site are even smaller, contradicting the explanation in the corresponding publication (Plaza-Garrido et al, 2020). Its authors used MAP_CHANNELS and the radius of gyration of the dye (4.6 A ˚), which underestimates the molecular dimensions, to analyze the accessibility of two sites in the crystal.…”

Protonation states of hen egg-white lysozyme observed using D/H contrast neutron crystallography

“…Although some densities corresponding to H atoms were observed in these X-ray Fourier maps, the H atoms were not included in the PDB files. HEWL structures in other crystal forms [PDB entries 7ave (0.98 A ˚, pH 4.5; Ramos, Laux, Haertlein, Boeri Erba et al, 2021), 2vb1 (0.65 A ˚, pH 4.7; Wang et al, 2007) and 7p6m (0.89 A ˚; pH 4.5; in the triclinic form and PDB entries 6f1o (0.96 A ˚; pH 4.5; Plaza-Garrido et al, 2018) and 6sye (0.97 A ˚; pH 7.0; Plaza-Garrido et al, 2020) in the orthorhombic form] included H atoms. All pH values were acidic except that for PDB entry 6sye.…”