The use of glycerol in crystallization of T7 RNA polymerase: Implications for the use of cosolvents in crystallizing flexible proteins

Preparation of crystalline T7 RNA polymerase (RNAP) from mother liquors using ammonium phosphate or ammonium sulfate as precipitants required the presence of at least 15% glycerol. This was shown to be due to a shift in the relative stability of the amorphous and crystalline phases of the enzyme in the presence of glycerol and was not a kinetic effect. Sucrose had effects similar to, but less dramatic than, those of glycerol. Since glycerol and polyhydric alcohols have been shown to have general stabilizing effects on protein conformation, we discuss the possibility that the T7 RNAP results indicate that glycerol and polyhydric alcohols may be generally useful in crystallizing proteins in which conformational flexibility is a problem. The mechanism of the glycerol effect is discussed in terms of modifications in protein hydration and an increased solvophobic effect.