[3] N-terminal modifications that alter P450 membrane targeting and function

This chapter discusses the N -terminal modifications that alter P450 membrane targeting and function. The hydrophobic N -terminal region that is present in mammalian P450 cytochromes serves as the signal peptide for cotranslational insertion of the protein into the endoplasmic reticulum, while the following hydrophobic proline-rich segment promotes stabilization of the enzyme. The role of the N -terminal region in promoting extensive aggregation of P450 enzymes is demonstrated by disaggregation of shortened P450s to monomers by the detergent cholate at concentrations that do not alter the multimeric aggregation state of the full-length enzymes. The retention of catalytic activity and the monomerization of P450 cytochromes at low detergent concentrations, achieved through N -terminal modification, are used to improve the suitability of these enzymes as candidates for crystallization attempts. The chapter describes the methods for the heterologous expression, determination of subcellular localization, and purification of shortened P450 cytochromes as well as examination of their catalytic properties.