LAUSR

Gaining structural information on membrane proteins in their native lipid environment is a long‐standing challenge in molecular biology. Instead, it is common to employ membrane mimetics, which has been shown to affect protein structure, dynamics, and function severely. Here, we describe the incorporation of a bacterial outer membrane protein (OmpW) into natively excreted membrane vesicles for solution nuclear magnetic resonance (NMR) spectroscopy using a mutant Escherichia coli strain with a high outer membrane vesicle (OMV) production rate. We collected NMR spectra from both vesicles containing overexpressed OmpW and vesicles from a control strain to account for the presence of physiologically relevant outer membrane proteins in vesicles and observed distinct resonance signals from OmpW. Due to the increased production of OMVs and the use of non‐uniform sampling techniques we were able to obtain high‐resolution 2D (HSQC) and 3D (HNCO) NMR spectra of our target protein inside its native lipid environment. While this workflow is not yet sufficient to achieve in situ structure determination, our results pave the way for further research on vesicle‐based solution NMR spectroscopy.