Removal of salt from a salt-induced protein crystal without cross-linking. Preliminary examination of "desalted" crystals of phosphoglucomutase by x-ray crystallography at low temperature

Abstract

A model procedure for removing salt from relatively fragile salt-induced protein crystals is proposed. The procedure is based on physical principles and is validated by using millimeter-size crystals of rabbit muscle phosphoglucomutase grown from a 2.1 M solution of ammonium sulfate. Three types of operations are included in the procedure: initial transfer to salt solutions of reduced concentration; transfer to the organic-rich phase of an equilibrium biphasic mixture obtained with aqueous solutions of polyoxyethylene and the salt; and addition of various replacement cosolutes in aqueous solutions of polyoxyethylene to reduce osmotic stress on the crystal as the remaining salt is removed. A critical feature of the overall procedure is maintenance of near equilibrium throughout by using a large number of steps involving small changes in solute concentration. The conditions used in the actual transfer were adjusted to eliminate the fracturing of crystals by visually distinguishing between two opposing types of fracture patterns: those produced by osmotic crushing as opposed to osmotic expansion. Basic requirements for a successful procedure with other protein crystals are a high permeability toward small solutes and a relatively slow dissolution rate at salt concentrations for which biphasic mixtures can be obtained. Desalted crystals of phosphoglucomutase have no visible fractures, are stable in the final solution for at least a week, and exhibit no noticeable change in the resolution of their X-ray diffraction pattern. In fact, desalted crystals can be rapidly cooled to 160 K, whereas untreated crystals are almost completely disordered by the same cooling procedure. The component of the desalting mixture whose presence is crucial to the success of the cooling process is polyoxyethylene, which apparently impedes the formation of ice within the protein crystal. Diffraction data obtained with an area-detector diffractometer did not differ significantly, either in terms of quality or resolution range, between crystals in 2.3 M ammonium sulfate at room temperature and crystals at 160 K in which ammonium sulfate had been replaced by glycine. The successful use of the following replacement solutes, instead of glycine, also is documented: sucrose, glycerol, and a low molecular weight poly(ethylene glycol) (PEG-400).

Extension of the diffraction resolution of crystals

“…"/,~ was observed. (Teng, 1990;Ray et al, 1991). Many have noted an increase in the crystal lifetime and an improvement in the diffraction resolution of crystals.…”

“…Another critical factor in the experiments was the 24-step transfer of crystals into the cryoprotective mixtures. Most recently, Ray et al (1991) demonstrated the importance of very gradual changes in the concentrations of additives to crystals to prevent internal osmotic shocks which caused irreversible damage. Because PEG was introduced by gradual transfer techniques, the solvent content was lowered by 6% without crystal damage.…”

“…To alter the diffraction properties, a procedure similar to that described by Ray et al (1991) was used. Each crystal was individually transferred, at 277 K, to a series of 24 droplets in Cryschem plates containing 50 ~al of decreasing concentrations of the equilibration solution and increasing concentrations of the cryoprotective mixtures listed in Table 1.…”

Soaking of crystals for macromolecular crystallography in a capillary

“…The process of the exchange of mother liquor sometimes leads to crystal cracking and/or significant loss of the X-ray scattering power of the crystals. This could in some cases be related to crystal crushing by osmotic pressure, especially when high-ionic-strength precipitant is exchanged for polyglycols (Ray, Puvathingal, Bolin, Minor, Liu & Muchmore, 1991). More often, however, crystal cracking is due to changes in the unit-cell dimensions that result from conformational adjustments induced by ligand binding to the protein molecules and subsequent changes in intermolecular contacts.…”

“…In some applications up to 40 exchanges of mother liquor were used (Ray et al 1991). It should be mentioned, however, that if the ligand binding is virtually irreversible then even very low concentrations of the ligand may lead to a large gradient of the protein-ligand complex in the crystals.…”