USE OF GLYCEROL, POLYOLS AND OTHER PROTEIN-STRUCTURE STABILIZING AGENTS IN PROTEIN CRYSTALLIZATION

A protein preparation to be used for crystallization should be homogen eous and should remain so throughout the course of a prolonged crystal lization 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 kno wn. Crystallographers are less aware of general methods for stabilizat ion of proteins against non-covalent modifications (partial denaturati on, heterogeneous aggregation) which can also introduce structural het erogeneity into a protein preparation. Related to this issue are metho ds to suppress protein conformational flexibility which can be a sourc e of dynamic structural heterogeneity and which presents an entropic b arrier to crystallization. However, for many years agents which stabil ize protein structure have been described in the biochemical literatur e. 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 crys talline proteins. Subsequently, glycerol was used with good effect in the crystallization of a number of proteins. Other recent results sugg est that soaking crystals in solutions containing glycerol can have 's tructure-ordering' effects on the crystalline protein. These observati ons support the utility of glycerol in protein crystallization and sug gest that the information in the biochemical literature on protein str ucture-stabilizing agents will find useful application in the field of protein crystal growth.