Probing Substrate-Loaded Carrier Proteins by Nuclear Magnetic Resonance.

Carrier proteins (CPs) are central actors in nonribosomal peptide synthetases (NRPSs) as they interact with all catalytic domains, and because they covalently hold the substrates and intermediates leading to the final product. Thus, how CPs and their partner domains recognize and engage with each other as a function of CP cargos is paramount to understanding and engineering NRPSs. However, rapid hydrolysis of the labile thioester bonds holding substrates challenges molecular and biophysical studies to determine the molecular mechanisms of domain recognition. In this chapter, we describe a protocol to counteract hydrolysis and study loaded carrier proteins at the atomic level with nuclear magnetic resonance (NMR) spectroscopy. The method relies on loading CPs in situ, with adenylation domains in the NMR tube, to reach substrate-loaded CPs at steady state. We describe controls and experimental readouts necessary to assess the integrity of the sample and maintain loading on CPs. Our approach provides a basis to conduct subsequent NMR experiments and obtain kinetic, thermodynamic, dynamic, and structural parameters of substrate-loaded CPs alone or in the presence of other domains.

Competing HB acceptors: an extensive NMR investigations corroborated by single crystal XRD and DFT calculations.

A series of N-benzoylanthranilamide derivatives have been synthesized with the substitution of competitive HB acceptors and investigated by NMR spectroscopy and single crystal XRD. The interesting rivalry for HB acceptance between [double bond splayed left]C[double bond, length as m-dash]O and X (F or OMe) is observed in the investigated molecules which leads to an unusual increase in the electron density at the site of one of the NH protons, reflecting in the high field resonance in the 1H NMR spectrum. The NMR experimental findings and single crystal XRD are further reinforced by the DFT studies.