Thermal contraction of aqueous glycerol and ethylene glycol solutions for optimized protein-crystal cryoprotection

Abstract

The thermal contraction of aqueous cryoprotectant solutions on cooling to cryogenic temperatures is of practical importance in protein cryocrystallography and in biological cryopreservation. In the former case, differential contraction on cooling of protein molecules and their lattice relative to that of the internal and surrounding solvent may lead to crystal damage and the degradation of crystal diffraction properties. Here, the amorphous phase densities of aqueous solutions of glycerol and ethylene glycol at T = 77 K have been determined. Densities with accuracies of <0.5% to concentrations as low as 30%(w/v) were determined by rapidly cooling drops with volumes as small as 70 pl, assessing their optical clarity and measuring their buoyancy in liquid nitrogen-argon solutions. The use of these densities in contraction matching of internal solvent to the available solvent spaces is complicated by several factors, most notably the exclusion of cryoprotectants from protein hydration shells and the expected deviation of the contraction behavior of hydration water from bulk water. The present methods and results will assist in developing rational approaches to cryoprotection and an understanding of solvent behavior in protein crystals.

The impact of cryosolution thermal contraction on proteins and protein crystals: volumes, conformation and order

“…The preferential exclusion of cosolvents from the protein surface may also play a role (Timasheff, 2002a,b;Auton et al, 2011;Shen et al, 2016). In comparison to water molecules, we observe five to ten times fewer cryoprotective agents in our electron-density maps than would be expected for concentrations of 50%(w/w) (Supplementary Tables S5, S7, S9 and S11).…”

“…Contractions for some solutions were interpolated or extrapolated as follows. For the glycerol solutions, the vitrified contraction values were crosschecked with those of Shen et al (2016) For four crystals, measurements were made to test the effects of the external cryoprotectant only by keeping a constant aqueous internal cryoprotectant and using different external oils with a range of contractions (11.3-5.0% contraction). For glycerol/water, MPD/water and DMSO/water solutions, Fig.…”

“…For example, Mitchell and Garman studied crystals of glycogen phosphorylase B (50% solvent, maximum pore radius 7.2 Å ), showing an optimum glycerol concentration for cryocooling of about 45%(w/v) (Mitchell & Garman, 1994) corresponding to a solvent contraction of about 4.5% (Shen et al, 2016), which falls in the above range. For example, Mitchell and Garman studied crystals of glycogen phosphorylase B (50% solvent, maximum pore radius 7.2 Å ), showing an optimum glycerol concentration for cryocooling of about 45%(w/v) (Mitchell & Garman, 1994) corresponding to a solvent contraction of about 4.5% (Shen et al, 2016), which falls in the above range.…”

“…Cooling is known to increase the true crystal mosaicity severalfold (Vahedi-Faridi et al, 2003). Information about the effects of the cryoprotective agent as well as concentration on contraction are available (Juers & Matthews, 2004a;Alcorn & Juers, 2010;Shen et al, 2016Shen et al, , 2017. More information may thus be uncovered by using a synchrotron-radiation source, especially without focusing optics, in which case mosaicity changes with cooling will represent a larger fraction of the base mosaicity (Bellamy et al, 2000) Cryo-optimization processes can then include knowledge about the cryosolvent thermal contraction.…”

“…Even when cooling conditions are found to be adequate for a particular purpose, they can often be further optimized by adjusting the concentration or the identity of the cryoprotective agent (Mitchell & Garman, 1994). The average contractions of many different cryosolutions between RT and 77 K have been measured both in macroscopic samples ($1 ml; Marshall et al, 2012;Juers & Matthews, 2001, 2004aAlcorn & Juers, 2010) and in microlitre-sized drops (Shen et al, 2016(Shen et al, , 2017, but systematic testing of contraction-based cryo-optimization has not yet been reported. The thermal behavior of such a system is complex, with potentially different thermal responses of the protein, internal solvent and external solvent.…”