METHODS: A Companion to Methods in Enzymology Vol. 1, No. I, August, pp. 50-56, 1990 The Use of Glycerol in Crystallization of T7 RNA Polymerase: Implications for the Use of Cosolvents in Crystallizing Flexible Proteins Rui Sousa 1 and Eileen M. Lafer Department of Biological Sciences, University of Pittsburgh, A524 Langley Hall, Pittsburgh, Pennsylvania 15260 Preparation of crystalline T7 RNA polymerase (RNAP) from mother liquors using ammonium phosphate or ammonium sul- fate as precipitants required the presence of at least 15% glyc- erol. 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 pro- teins in which conformational flexibility is a problem. The mech- anism of the glycerol effect is discussed in terms of modifica- tions in protein hydration and an increased solvophobic effect. © 1990Academic Press, Inc. Construction of a well-ordered protein crystal lattice results not only in the loss of the rotational and transla- tional entropy associated with the freedom of movement of the protein as a whole in solution but also in the loss of entropy associated with the movement of individual atoms and groups of atoms within the protein. In other words, construction of a well-ordered crystal lattice places constraints on the degree of conformational flex- ibility accessible to a protein. By comparison, the con- straints on conformational flexibility in an amorphous precipitate will be relatively relaxed. One energetic bar- rier between crystallization and amorphous precipita- tion will therefore be the unfavorable entropy associated with loss of conformational flexibility when the protein is restricted in a crystal lattice. Such an unfavorable en- tropy term will be larger when the extent of conforma- tional flexibility in a specific protein is large. Crystalliza- tion of flexible proteins depends on the existence of other favorable energetic terms, i.e., particularly favor- able intermolecular contacts in the crystal lattice, which i To whom correspondence should be addressed. 50 can overcome this unfavorable entropy term. When the unfavorable entropy associated with the loss of confor- mational freedom in the crystalline versus amorphous state is too large relative to the favorable energy terms associated with crystallization, the amorphous state will be thermodynamically more stable than the crystalline state and crystallization will be impossible. Practically, one can seek to decrease the size of the unfavorable entropy term by screening for solution con- ditions (pH, etc.) or ligands that suppress protein con- formational flexibility. A ligand may bind to the protein and thereby specifically stabilize one conformation. Be- cause such ligands, effector molecules, or solution envi- ronments are usually specific for particular proteins, identification of these agents in a specific case is rarely applicable to the crystallization of distinct proteins. It would be useful to identify an agent(s) that would invari- ably or frequently serve to suppress conformational flexibility. In seeking to identify such an agent it is useful to con- sider the processes of protein dissolution, enhancement of protein conformational flexibility, and protein dena- turation to be thermodynamically continuous. From this point of view one can classify protein folding and sup- pression of conformational flexibility as intramolecular precipitation, while what is usually called protein precip- itation is specified as intermolecular precipitation. Pow- erful denaturants such as urea and guanididium are also powerful protein solubilizing agents. Water, the primary solvating agent in a typical mother liquor used for crys- tallization, is not generally considered a denaturant, but experimental evidence has clearly demonstrated that de- creasing the amount of water available for interaction with a protein suppresses conformational flexibility and stabilizes proteins against denaturation (reviewed in (1)). For example, myelin basic protein has little or no secondary structure and no specific conformation in aqueous solution and has therefore been deemed "un- crystallizable" (2), but when myelin basic protein is en- closed in a reversed micelle environment it shows a dra- matic increase in CD-measurable secondary structure, 1046-2023/90 $3.00 Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.