In-Cell DEER Spectroscopy of Nanodisc-Delivered Membrane Proteins in Living Cell Membranes

Membrane proteins are integral to numerous cellular processes, yet their conformational dynamics in native environments remains difficult to study. This study introduces a nanodelivery method using nanodiscs to transport spin-labeled membrane proteins into the membranes of living cells, enabling direct in-cell double electron-electron resonance (DEER) spectroscopy measurements. We investigated the membrane protein BsYetJ, incorporating spin labels at key positions to monitor conformational changes. Our findings demonstrate successful delivery and high-quality DEER data for BsYetJ in both Gram-negative