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 μm) generated by Staphylococcus aureus sortase A and its peptide substrate under a real-time reaction have been assigned.