Carrier proteins (CPs) play a central role in nonribosomal peptide synthetases (NRPSs) as they shuttle covalently attached substrates between active sites. Understanding how the covalent attachment of a substrate (loading) influences the molecular properties of CPs is key to determining the mechanism of NRPS synthesis. However, structural studies have been impaired by substrate hydrolysis. Here, we used nuclear magnetic resonance spectroscopy to monitor substrate loading of a CP and to overcome hydrolysis. Our results reveal the spectroscopic signature of substrate loading and provide evidence of molecular communication between an NRPS carrier protein and its covalently attached substrate.