Strategy to Select an Appropriate Cryoprotectant for an X-ray Study of Escherichia coli GAPDH Crystals

The treatment of protein crystals with an appropriate cryoprotectant and immediate freezing in liquid nitrogen before data collection at the synchrotron source is one of the critical steps for determining the structure of three-dimensional biomacromolecule (protein) crystals. Three-dimensional crystal structures of glyceraldehyde 3-phosphate dehydrogenase (GAPDH: EC 1.2.1.12) have been determined in many species, including Escherichia coli (Ec), using crystallization and cryoprotectant agents. Herein, we describe the cryogenic effects on E. coli GAPDH (EcGAPDH) crystals that were crystallized with 2.8 M ammonium sulfate as a precipitating agent. Fully grown EcGAPDH crystals were frozen under various cryoprotection treatments, including 11 different cryoprotectants with different protein mechanisms. We found that the lattices of some EcGAPDH crystals stabilized well with 3.5 M ammonium sulfate, which is not typically used as a cryoprotectant because it does not have antifreeze properties. The observed stabilization may be due to an osmotic effect induced by the difference in concentration between the cryoprotectant (3.5 M) and the precipitating agent (2.8 M), which may lead to dehydration of the inside of the EcGAPDH crystals. These findings suggest that the use of ammonium sulfate at a certain concentration (3.5 M), higher than the precipitating agent, may be a useful cryoprotectant for other protein crystals. Additionally, although the addition of salts with antifreezing properties such as lithium sulfate (1 M) or sodium formate (3 M) to the crystallization condition of EcGAPDH (2.8 M ammonium sulfate) successfully provided cryoprotection, it did not yield improved X-ray data compared to crystals of EcGAPDH cryoprotected with 3.5 M ammonium sulfate or trehalose.