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Nonribosomal peptide synthetases (NRPSs) are microbial enzymatic systems that synthesize a wealth of important natural products such as virulence factors (e.g. yersiniabactin) or antibiotics (penicillin, vancomycin). NRPSs employ conserved domains to tether simple substrates via thioester bonds and condense them into complex natural products via peptide bonds. Recent studies showed that NRPSs are not rigid but undergo a series of sequential, transient molecular interactions during synthesis. Nuclear magnetic resonance (NMR) is ideally suited for studies of such dynamic proteins and for characterizing transient interactions, but the method is often limited to proteins smaller than many NRPS domains. Here, we showcase the molecular information provided by NMR with studies of small NRPS carrier proteins, and we describe NMR methods to overcome challenges in studies of larger proteins.

We present the first structure of an NRPS carrier protein covalently attached to its substrate via a labile thioester bond. We show that the substrate interacts with the protein core, albeit in a transient manner. This interaction modulates protein dynamics and alters surfaces involved in molecular binding. Our observations provide a molecular basis for interplay between the successive chemical reactions that occur in NRPS synthesis and related domain communication.

NMR resonances assignment is a major bottle neck for studies of larger proteins.聽 To assign NMR signals, investigators must analyze a series of spectra and identify signals that belong to sequential residues, and this protocol becomes exponentially more difficult as the number of residues increases. We have translated this procedure into mathematical operations that can be applied to NMR spectra. Thus, a single correlation map harnesses the information of all data collected, and resonance assignment is obtained by visual inspection. The method is illustrated for assignment of backbone and side-chain resonances of 37 kDa and 52 kDa NRPS domains.