3h adomet

MTase reactions with purified recombinant proteins were performed at 37°C for 1 h in 50 μl reactions containing assay buffer [50 mM Tris (pH 7.8 at 25°C), 50 mM KCl, 5mM MgCl₂, 1mM ATP, 13 μM [³H]-AdoMet (Perkin Elmer)], 4 μM substrate and 2 μM MTase unless noted otherwise. Reactions were terminated by adding 900 μl ice cold TCA and precipitated material was isolated on a glass fibre filter (Whatman GF/C) using vacuum filtration. Filters were then washed with TCA and absolute ethanol, and incorporated methyl groups were quantified by scintillation counting. For MS analysis radiolabeled AdoMet was replaced by 1.2 mM unlabeled AdoMet (NEB) and MTase reactions were terminated by boiling in NuPAGE buffer (Life Technologies).
MTase reactions with protein extracts as source of enzymatic activity and substrate were performed in the same assay buffer as described above but were instead incubated at 30°C for 1 h. As source of enzymatic activity, 50 μg of a protein extract from E. coli overexpressing 6xHis-Ynl024c was used, and 50 μg of protein extract from the ynl024cΔ yeast strain was used as a source of substrate(s). Proteins were then separated by SDS-PAGE and transferred to a PVDF membrane. The membrane was stained with Ponceau S, treated with the scintillation enhancer EN³HANCE and exposed to Carestream Kodak BioMax MS film (Sigma-Aldrich) for 8 weeks at -80°C.

MTase activity assay using C. elegans MT13-N or MT13-C as enzymes, human eEF1A1 as the substrate and [³H]AdoMet (PerkinElmer) as the methyl donor, was performed similarly as previously described [24 (link)]. 10 μl reactions were set-up on ice in Storage Buffer, containing 1 μg of eEF1A1, 1 μg of MT13-N or MT13-C and 0.5 μCi of [³H]AdoMet. Reaction mixtures were incubated for 1 h at 30°C and analyzed by SDS-PAGE. Proteins were transferred to polyvinylidene difluoride membrane and stained with Ponceau S. Incorporation of tritium-labeled methyl groups into proteins was visualized by fluorography. All methyltransferase experiments were independently replicated at least two times.

Methylation assays were performed as previously described by Schmidt et al. (2011 (link)). Methylation assays of the MCPs by CheR1 were performed by measuring the transfer of radioactivity from the methyl donor [³H] Ado-Met SAM to MCP-containing membranes fraction. A reaction mixture (100 μl) containing 50 μl of MCP-containing membrane fraction and 0.1 μM purified 6 × His-CheR1 in reaction buffer [50 mM NaH₂PO₄ (pH 8.0); 300 mM NaCl)] was pre-incubated at 30°C for 10 min, and then 3 μl 0.625 μM [³H] Ado-Met (specific activity, 15 Ci/mmol; PerkinElmer) was added to the reaction mixture to initate methyl transfer reaction. After incubating at 30°C for 30 min the methylation reaction was stopped by adding 2 × SDS-PAGE loading dye. The reaction products were subjected to SDS-PAGE (12% polyacrylamide), and ³H-labeled MCP proteins were then visualized by autoradiography in each reaction. The effect of CheR1 (0.1 μM), MapZ (0.1 μM), c-di-GMP (10 μM) on methylation was examined by incubating the protein and c-di-GMP in the reaction mixture accordingly.

Methyltransferase assays were performed in 40 mM Tris-HCl (pH 8.0), 5 mM DTT, 2 μM ^7MeGpppA or GpppA RNA cap analogue (New England Biolabs), 10 μM adenosyl-methionine (AdoMet; Thermo Fisher), 0.03 μCi/μl [³H]AdoMet (PerkinElmer) (25 (link)). For each reaction, MERS-CoV or SARS-CoV nsp14 was added to a final concentration of 500 or 250 nM, respectively. Reaction mixtures were incubated at 30°C for up to 120 min, and reactions were stopped by the addition of a 10-fold volume of 100 μM ice-cold adenosyl-homocysteine (AdoHcy; Thermo Fisher). Then, samples were spotted on a DEAE filter mat (PerkinElmer) prewet with Tris-HCl (pH 8.0) buffer. Filter mats were washed twice with 10 mM ammonium formate (Sigma-Aldrich) (pH 8.0), twice with MilliQ water, and once with absolute ethanol (Sigma-Aldrich). After air drying for 10 min, filter mats were cut, and relevant pieces were transferred to individual tubes. Betaplate scintillation fluid (PerkinElmer) was added, and the amount of ³H label bound was measured in counts per minute using a Wallac scintillation counter. For relative quantification, incorporation measurements for mutant proteins were normalized to values obtained with the wt control nsp14. Samples were measured in duplicate in each experiment.

PRMT activity assays were analyzed by the incorporation of [³H] from [³H] AdoMet to histones (HMT assays) as described [25 ]. The EhPRMT1a recombinant protein (1 μg) was incubated with 10 μg of the nuclear basic fraction of E. histolytica or with 0 to 4 μg of histones from chicken erythrocytes (Millipore) and 1 μCi of [³H] AdoMet (PerkinElmer Life Sciences) in HMT buffer (50 mM Tris–HCl pH8.0, 5% glycerol, 0.1 mM EDTA, 50 mM KCl, 1 mM DTT, 10 mM sodium butyrate) and in the presence of a protease inhibitor cocktail (Sigma P2714), in a reaction volume of 10 μl. After incubation of 45 min at 30°C, reactions were spotted onto P81 filter circles (Whatman) and allowed to air dry. Then, filters were washed three times with trichloroacetic acid for 15 min, rinsed in ethanol and allowed to air dry. Finally, radioactivity in filters was counted in 4 ml of scintillation fluid (Ready Safe, Beckman Coulter) in a scintillation counter (Beckman LS 6500). These assays were performed three times by duplicate. On the other hand, to determine whether EhPRMT1a methylates the H4R3 residue, we performed HMT assays using reactions containing a mixture of commercial histones (4 μg), non-radioactive AdoMet and 1 μg of the recombinant EhPRMT1a. As a control we performed the reaction in the presence of 1 μg of lysozyme. Finally, reactions were analyzed by Western blot using the α-H4R3me2 antibody.