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Protein and peptide prenylation is an essential biological process involved in many signal transduction pathways. Hence, it plays a critical role in establishing many major human ailments, including Alzheimer's disease, amyotrophic lateral sclerosis (ALS), malaria, and Ras-related cancers. Yeast mating pheromone a-factor is a small dodecameric peptide that undergoes prenylation and subsequent processing in a manner identical to larger proteins. Due to its small size in addition to its well-characterized behavior in yeast, a-factor is an attractive model system to study the prenylation pathway. Traditionally, chemical synthesis and characterization of a-factor have been challenging, which has limited its use in prenylation studies. In this chapter, a robust method for the synthesis of a-factor is presented along with a description of the characterization of the peptide using MALDI and NMR. Finally, complete assignments of resonances from the isoprenoid moiety and a-factor from COSY, TOCSY, HSQC, and long-range HMBC NMR spectra are presented. This methodology should be useful for the synthesis and characterization of other mature prenylated peptides and proteins. 

The integral membrane protease ZMPSTE24 plays an important role in the lamin A maturation pathway. ZMPSTE24 is the only known enzyme to cleave the last 15 residues from the C-terminus of prelamin A, including a farnesylated and carboxyl methylated cysteine. Mutations in ZMPSTE24 lead to progeroid diseases with abnormal prelamin A accumulation in the nucleus. Ste24 is the yeast functional homolog of ZMPSTE24 and similarly cleaves the a-factor pheromone precursor during its posttranslational maturation. To complement established qualitative techniques used to detect the upstream enzymatic cleavage by ZMPSTE24 and Ste24, including gel-shift assays and mass spectrometry analyses, we developed an enzymatic in vitro FRET-based assay to quantitatively measure the upstream cleavage activities of these two enzymes. This assay uses either purified enzyme or enzyme in crude membrane preparations and a 33-amino acid a-factor analog peptide that is a substrate for both Ste24 and ZMPSTE24. This peptide contains a fluorophore (2-aminobenzoic acid—Abz) at its N-terminus and a quencher moiety (dinitrophenol— DNP) positioned four residues downstream from the cleavage site. Upon cleavage, a fluorescent signal is generated in real time at 420 nm that is proportional to cleavage of the peptide and these kinetic data are used to quantify activity. This assay should provide a useful tool for kinetic analysis and for studying the catalytic mechanism of both ZMPSTE24 and Ste24.