Purification and crystallization of N-terminally truncated forms of microsomal cytochrome P450 2C5

This chapter describes the preparation and crystallization of the enzyme and discusses procedures contributing to the improved resolution of the crystals that should be applicable to other modified membrane P450s. Engineering more soluble forms of P450 2C5 has contributed to the crystallization of the enzyme. When detergents are used in both crystallization and purification of the protein, the ability to control the content and identity of the detergent is dependent on the protein exhibiting a sufficient degree of solubility to permit its concentration in the absence of detergents. The production of concentrated solutions of the protein containing little or no detergent provides a means for screening crystallization conditions and the selection of detergents that facilitate crystallization. These detergents can then be used to improve the purification of the protein and to solubilize substrates for the cocrystallization of enzyme-substrate complexes. In addition, the protein is sufficiently soluble to purify the enzyme without the use of detergents. Substitution of five amino acid residues derived from a related enzyme, P450 2C3, for the corresponding residues in P450 2C5 further increased the solubility and monodispersity of the protein, P450 2C5/3LVdH, relative to that of P450 2C5dH.