Use of glycerol, polyols and other protein structure stabilizing agents in protein crystallization

A protein preparation to be used for crystallization should be homogeneous and should remain so throughout the course of a prolonged crystallization experiment. General methods for preparation of pure proteins and for prevention of their covalent modification (through proteolysis, sulfhydryl oxidation, etc.) during prolonged incubation are well known. Crystallographers are less aware of general methods for stabilization of proteins against non-covalent modifications (partial denaturation, heterogeneous aggregation) which can also introduce structural heterogeneity into a protein preparation. Related to this issue are methods to suppress protein conformational flexibility which can be a source of dynamic structural heterogeneity and which presents an entropic barrier to crystallization. However, for many years agents which stabilize protein structure have been described in the biochemical literature. Recently the most widely used of these structure-stabilizing agents, glycerol, was used to crystallize T7 RNA polymerase. The observation that this compound has general structure-stabilizing effects and that it was essential for crystallization of at least this one protein led to the suggestion that it might be generally useful in crystallizing flexible proteins and in inducing order in disordered segments of crystalline proteins. Subsequently, glycerol was used with good effect in the crystallization of a number of proteins. Other recent results suggest that soaking crystals in solutions containing glycerol can have 'structure-ordering' effects on the crystalline protein. These observations support the utility of glycerol in protein crystallization and suggest that the information in the biochemical literature on protein structure-stabilizing agents will find useful application in the field of protein crystal growth.