Solid-phase synthesis of C-terminal peptide libraries for studying the specificity of protein farnesyltransferase

Protein prenylation is a common post-translational modification of specific protein-derived cysteine residues in eukaryotic cells. To study the substrate specificity of these enzymes, the primary strategy employed to date has involved the synthesis, purification and assaying of individual peptides. As an improvement, here we describe the synthesis of peptides containing free C-termini on solid supports. The libraries were screened using an alkyne-containing isoprenoid analogue followed by click chemistry with biotin azide and subsequent visualization with streptavidin-AP. Screening of the CVa2X and CCa2X libraries with R. norvegicus PFTase revealed reaction by many known recognition sequences as well as numerous unknown ones. Screening of the CVa2X library with alkyne-functionalized isoprenoid substrates showed that those prepared from C10 or C15 precursors gave similar results while the analogue synthesized from a C5 unit gave a different pattern of reactivity. Finally the substrate specificities of PFTases from three organisms (R. norvegicus, S. cerevisiae and C. albicans) were compared using CVa2X libraries. R. norvegicus PFTase was found to share more peptide substrates with S. cerevisiae PFTase than with C. albicans PFTase. In general, this method is a highly efficient strategy for rapidly probing the specificity of this important enzyme.