Chemistry Seminar with Dr. Francesca M. Marassi from the Medical College of Wisconsin at 4:00pm

Abstract: Understanding how protein structure and function relate to the biological environment is critical for gaining mechanistic insights. Nuclear magnetic resonance (NMR) is exceptionally well suited for this purpose because NMR signals are highly susceptible to the local environment and capable of reporting even very weak intermolecular interactions. Here, we show how NMR experiments can be performed directly on membrane proteins that are natively incorporated in the extracellular outer membrane vesicles (OMV) produced by Gram-negative bacterial cells. Bacterial OMVs play key roles in cell envelope homeostasis, secretion, interbacterial communication, and pathogenesis. In the case of the facultative intracellular pathogen Salmonella Typhimurium, OMV production is increased inside the acidic vacuoles of host cells by upregulating the expression of the outer membrane protein PagC. Recently, we identified a pH-responsive amino acid motif in the extracellular loops of PagC that regulates OMV production, and here we present NMR data for PagC natively incorporated in bacterial OMVs. The data, combined with molecular dynamics (MD) simulations, electron microscopy (EM), and microbial activity assays, support a mechanism where protonation of key histidine residues in the extracellular loops of PagC leads to changes in protein structure, flexibility and interactions with the surrounding outer membrane lipids. The data point to a mechanism for sensing and responding to environmental pH and for outer membrane protein control of membrane dynamics. The study underscores the unique power of NMR to examine protein structure and interactions in native biological contexts.