Chemical Structure from Scientific Research

RP-HPLC (280 nm; 15 min 20-50% acetonitrile gradient) analysis of the ligation of a model NGL H-containing acyl donor peptide (Ac-RWRGWR NGL H, 0.1 mM) to Lys-containing acyl acceptor peptides (0.5 mM) as catalysed by 1 M Oa AEP1 in HEPES buffer containing 0.2 mM NiSO 4 (pH 8.5) after 1.5 h at 25 C ( n = 3 independent experiments with similar results; representative data shown). Relative peak areas were used to calculate conversion of the limiting Ac-RWRGWR NGL H peptide to the isopeptide-linked product. The hydrolysis product is Ac-RWRGWRN-OH. We note that peaks for the Lys-containing acyl acceptor peptides are not detected at 280 nm. See Supplementary Fig. 1 for MALDI-TOF MS spectra of these reactions and Supplementary Figs. 2 and 3 for RP-HPLC and MALDI-TOF MS analysis of equivalent reactions without NiSO 4.

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