Extended Data Fig. 10: Determination of the necessity of polar residues in the precursor peptide for Lan formation.
From: Pseudokinases can catalyse peptide cyclization through thioether crosslinking
(a) Precursor peptide SacA. The mutation sites in the precursor peptide are indicated by asterisks. (b) Analysis of deuterium exchange through enzymatic ring opening and recylization in D2O. (i), treatment of 6E30A (top, control) with SacE (bottom); (ii), treatment of 6E33A (top, control) with SacE (bottom); and (iii), treatment of 6D53A (top, control) with SacE (bottom) in the mixture containing 50 mM Tris-HCl (pH 7.5). (c) Analysis of 7 variant production based on NEM derivatization. (i), treatment of 6E30A (top, control) with SacE (bottom); (ii), treatment of 6E33A (top, control) with SacE (bottom); and (iii), treatment of 6D53A (top, control) with SacE (bottom) in the presence of NEM. (d) Analysis of deuterium exchange of 6D19A under basic conditions. Samples include 6D19A (top, control) and the mixture of 6D19A (top, control) with SacE (bottom) and 50 mM Tris-HCl (pH 9.0) in D2O. (e) Proposed mechanism of D19-involved thiol deprotonation during retro-Michael and Michael reactions. For panels b, c and d, related experiments were performed >= 3 times (with >= 2 parallel samples for each).
