Dansyl gcvls

FTase and GGTase I activity was determined using the method of Temple et al.,^{39 (link)} with some modifications. For FTase, reaction mixtures were initiated by the addition of 18 nM recombinant FTase (Jena Biosciences) to reaction buffer (50 mM HEPPSO, 5 mM TCEP-HCl, 5 mM MgCl₂) containing 10 μM FDP (Sigma) and 5 μM dansyl-GCVLS (Biosynthesis, Inc.). For GGTase I, 24 nM of recombinant GGTase I (Jena Biosciences) was added to reaction buffer (50 mM HEPPSO, 5 mM TCEP-HCl,) containing 10 μM GGDP (Sigma) and 5 μM dansyl-GCVLS (Biosynthesis, Inc.). Reactions were carried out at 30 °C in the presence or absence of test compounds for 1 hour (FTase) or 2 hours (GGTase I). Prenylation of the peptide results in an increase in fluorescence intensity of the dansyl group (λ_ex = 340 nm, λ_em = 520 nm). Changes in fluorescence over time were detected using a Molecular Devices Spectramax Gemini EM fluorescence microplate reader.

FTase and GGTase I activity was determined using the methods of Cassidy et al.^{30 (link)} and Pickett et al.^{31 (link)} with some modifications. For FTase, reaction mixtures were initiated by the addition of 3 nM recombinant FTase (Jena Biosciences) to reaction buffer (50 mM Tris-HCl, pH 7.5, 5 mM DTT, 10 mM MgCl₂, 10 µM ZnCl₂, 0.04% n-dodecyl β-d-maltoside (DDM)) containing 10 µM FPP and 5 µM dansyl-GCVLS (Biosynthesis, Inc.). For GGTase I, 12 nM of recombinant GGTase I (Jena Biosciences) was added to reaction buffer (50 mM Tris-HCl, pH 7.5, 5 mM DTT, 5 mM MgCl₂, 50 µM ZnCl₂, 10 mM KCl, 0.04% DDM) containing 10 µM GGPP and 7 µM dansyl-GCVLS (Biosynthesis, Inc.). Reactions were carried out at 30 °C in the presence or absence of test compounds. After one hour, reaction mixtures were placed on ice, and the reaction was stopped by the addition of acetonitrile containing 5% HCl. The mixture was briefly vortexed and then spun at 14000 rpm for 5 minutes. 100 µL of the supernatant was injected into an HPLC with fluorescence detection and the prenylated fluorescent peptides were quantified.

FTase and GGTase I activity was determined using the method of Temple et al.,^{48 (link)} with modifications as previously described.^{22 (link)} Recombinant FTase and GGTase I were obtained from Jena Biosciences and dansyl-GCVLS and dansyl-GCVLL were obtained from Biosynthesis, Inc. Changes in fluorescence over time were detected using a Molecular Devices Spectramax Gemini EM fluorescence microplate reader. Compounds were tested in duplicate at multiple concentrations and three independent experiments were performed.