Resonance Assignments of Lowly Populated and Unstable Enzyme Intermediate Complex under Real‐Time Conditions

Unstable and low‐abundance protein complexes represent a large family of transient protein complexes that are difficult to characterize, even by means of high‐resolution NMR spectroscopy. A method to assign the NMR signals of these unstable complexes through a combination of selective isotope labeling of amino acids in a protein and site‐specific labeling the protein with a paramagnetic tag is presented herein. By using this method, the resonances of unstable thioester intermediate complex (lifetime <5 h and highest concentration ≈20 μ m ) generated by Staphylococcus aureus sortase A and its peptide substrate under a real‐time reaction have been assigned.