BPS2026 – A strategy to trap the denatured state of a helical-bundle membrane protein in lipid bilayers

Understanding denatured states of proteins is crucial as they determine thermodynamic stability of proteins with native states, serve as the initial state in the folding mechanisms, and are targets for protein turnover, chaperone action, and translocation. Compared to water-soluble proteins, the conformational features of the denatured state ensembles (DSEs) of membrane proteins are largely unexplored. A major challenge lies in reconstituting a realistic DSE model under native conditions for biophysical characterization. We previously developed such a model using the steric trapping strategy (Gaffney et al. 2022 PNAS ). However, caveats here are that the sterically trapped DSE can be artificially expanded due to the repulsion between large protein beads and avert potential topology dynamics of transmembrane segments across the membrane. Here, we have established a new strategy to trap the DSE of a helical membrane protein, E. coli GlpG. This strategy exploits the conjugation of a small-molecule folding inhibitor, which reversibly traps the DSE by preventing the formation of native packing interactions. The conformational dynamics and compactness of the DSE are characterized using proteolysis and DEER-EPR, respectively. The reversible trapping of the DSE further allows us to investigate various physical properties of the lipid bilayer, including membrane thickness, fluidity, and lateral stress, on the folding efficiency of GlpG. This study will provide an understanding of the DSE of membrane proteins and the identification of environmental and cellular factors that affect the foldability of membrane proteins in lipid bilayers.