Abstract Biochemical processes within the living cell occur in a highly crowded environment. The phenomenon of macromolecular crowding is not an exclusive feature of the cytoplasm and can be observed in the densely protein-packed, nonhomogeneous cellular membranes and at the membrane interfaces. Crowding affects diffusional and conformational dynamics of proteins within the lipid bilayer, and modulates the membrane organization. However, the non-invasive quantification of the membrane crowding is not trivial. Here, we developed the genetically- encoded fluorescence-based sensor for probing the macromolecular crowding at the membrane interfaces. Two sensor variants, both composed of fluorescent proteins and a membrane anchor, but differing by the flexible linker domains were characterized in vitro , and the procedures for the membrane reconstitution were established. Lateral pressure induced by membrane-tethered synthetic and protein crowders altered the sensors’ conformation, causing increase in the intramolecular Förster’s resonance energy transfer. The effect of protein crowders only weakly correlated with their molecular weight, suggesting that other factors, such as shape and charge play role in the quinary interactions. Upon their expression, the designed sensors were localized to the inner membrane of E. coli , and measurements performed in extracted membrane vesicles revealed low level of interfacial crowding. The sensors offer broad opportunities to study interfacial crowding in a complex environment of native membranes, and thus add to the toolbox of methods for studying membrane dynamics and proteostasis.

Probing macromolecular crowding at the lipid membrane interface with genetically-encoded sensors