Soaking: the effect of osmotic shock on tetragonal lysozyme crystals

Abstract

Protein crystals crack when they are soaked in a solution with ionic strength suf®ciently different from the environment in which they grew. It is demonstrated for the case of tetragonal lysozyme that the forces involved and the mechanisms that lead to the formation of cracks are different for hypertonic and hypotonic soaking. Tetragonal lysozyme crystals are very sensitive to hypotonic shocks and, after a certain waiting time, cracks always appear with a characteristic pattern perpendicular to the crystallographic c axis. Conversely, a hypertonic shock is better withstood: cracks do not display any deterministic pattern, are only visible at higher differences in ionic strength and after a certain time a phenomenon of crystal reconstruction occurs and the cracks vanish. At the lattice level, the unit-cell volume expands in hypotonic shock and shrinks under hypertonic conditions. However, the compression of the unit cell is anisotropic: the c axis is compressed to a minimum, beyond which it expands despite the unit-cell volume continuing to shrink. This behaviour is a direct consequence of the positive charge that the crystals bear and the existence of channels along the crystallographic c axis. Both features are responsible for the Gibbs±Donnan effect which limits the free exchange of ions and affects the movement of water inside the channels and bound to the protein.

Humidity control and hydrophilic glue coating applied to mounted protein crystals improves X-ray diffraction experiments

“…However, glue-coated lysozyme crystals showed a different response to that of the uncoated crystals to humidity changes (Dobrianov et al, 2001;Salunke et al, 1985;Kodandapani et al, 1990;Kiefersauer et al, 2000), in which a large change in lattice constants (maximally 10%) and hysteretic responses were observed. Instead, our results are similar to the observation reported in soaking experiments with a higher concentration of sodium chloride (Ló pez-Jaramillo et al, 2002); that is, the response was small (less than a 1% change in lattice constants), the c axis expanded after dehydration and a hysteretic response was not observed. The response to humidity changes was reproducible regardless of the crystal orientation, which is different from the experiments with the uncoated crystals.…”

A Technique for High-Throughput Protein Crystallization in Ionically Cross-Linked Polysaccharide Gel Beads for X-Ray Diffraction Experiments

“…In most unsuccessful ligand-soaking experiments, some cracking or fragmentation of soaked crystals is observed [43]. During ligand soaking of highly sensitive crystals, formation of a multilayer poly-ion complex that completely covers the gel-bead surface may decrease the permeability of ligands into the crystals.…”

Gentle, fast and effective crystal soaking by acoustic dispensing

“…2), opens up possibilities for crystal-soaking experiments that are not possible to perform by hand. On the other hand, the compound is dispensed directly from 100% stock solutions, and sudden additions of solvent-altering components are in general stressful for protein crystals owing to osmotic effects (Ló pez-Jaramillo et al, 2002), and possibly also owing to mechanical stress as a results of the high-frequency (200 Hz) pulses of solvent droplets. We therefore investigated how the positional precision of acoustic transfer could be exploited to ensure that soaking was not only rapid but also sufficiently gentle.…”

“…Typical crystal-soaking experiments usually involve preparing the compound of interest in a crystal-compatible solution, usually reservoir solution with an additional solvent such as DMSO, after which either the solution is transferred directly to the crystallization drop or the crystal is moved to the solution. This exposes the crystal to sudden changes from its native solution, which can damage the crystal through osmotic shock (Ló pez-Jaramillo et al, 2002). Some crystals are far more tolerant to this form of treatment than others, but careful stepwise procedures can overcome this for those that are not, enabling higher solvent and compound concentrations to be introduced gradually to the crystal (Hassell et al, 2007).…”