Fig. 4: Investigation of OAZ NeissLock reaction.
From: NeissLock provides an inducible protein anhydride for covalent targeting of endogenous proteins
a Specific reaction by NeissLock. OAZ-GSY-SPM was incubated with Ca2+ for 16 h with ODC or non-cognate MBP or sfGFP (each protein at 10 µM). Samples were analyzed by SDS-PAGE with Coomassie staining. b Decreasing OAZ/ODC binding affinity blocked conjugation. Wild-type (wt) or point mutants of OAZ-GSY-SPM were incubated ± Ca2+ with ODC for 60 min with each protein at 0.5 µM, before SDS-PAGE with Coomassie staining. c Neighboring lysines on ODC. ODC/OAZ complex (PDB 4zgy) with Ct and nearby lysines (orange) shown in stick format. d Different sites on ODC reacted with OAZ. Potential crosslinking sites were removed from ODC to give ∆N (N-terminus truncated), ∆C (C-terminus truncated), and 4KR (K74R, K78R, K92R, K121R). Lysine was individually re-introduced at K92. OAZ-GSY-SPM was incubated with the indicated ODC mutant for 16 h at 37 °C ± Ca2+, before SDS-PAGE with Coomassie staining. Relative coupling compared to wt was quantified (mean of triplicate ± 1 s.d.). e Efficient NeissLock coupling to AZI. 2.5 µM AZI was incubated with 2.5 µM OAZ-GSY-SPM for 18 h at 37 °C ± Ca2+, before SDS-PAGE with Coomassie staining. f Rapid AZI coupling. 2.5 µM AZI was incubated with 2.5 µM OAZ-GSY-SPM in HEPES buffer, pH 7.4 at 37 °C with Ca2+. Coupling to AZI was quantified from SDS-PAGE with Coomassie (mean of triplicate ± 1 s.d.). Molecular weight markers represent kDa. Source data are provided as a Source Data file.
