Cellular membranes are densely packed with proteins and lipids, and are constantly remodeled by mechanical forces. Steric pressure generated by these forces is thought to play a significant role in key physiological processes, such as shaping endocytic vesicles and cellular protrusions. However, no method currently exists for measuring steric pressure at membrane surfaces. Recently we developed a sensor based on Förster resonance energy transfer (FRET), which uses the principles of polymer physics to precisely detect changes in steric pressure. The sensor consists of a polyethylene glycol chain tethered to the membrane surface. The polymer has a donor fluorophore at its free end, such that FRET with acceptor fluorophores in the membrane provides a real-time readout of polymer extension (Fig. A). As a demonstration of the sensor, we measured the steric pressure generated by a model protein involved in membrane bending, the N-terminal homology domain (ENTH) of Epsin1. As the membrane becomes crowded by ENTH proteins, the polymer chain extends, increasing the fluorescence lifetime of the donor (Fig. B, C). Having established the sensor, we compared the wild type ENTH protein, which binds to the membrane by inserting an amphipathic helix, to a histidine tagged mutant, which uses the artificial histidine-nickel interaction to bind the membrane (Fig. D). Drawing on polymer theory, we used a change in FRET lifetime to calculate steric pressure as a function of membrane coverage by ENTH, validating theoretical equations of state (Fig. E, F). Further, we found that ENTH’s ability to break up larger vesicles into smaller ones correlated with steric pressure rather than the chemistry used to attach ENTH to the membrane (Fig. G). This result demonstrates the potential for this sensor to measure steric pressure in situ during diverse biochemical events that occur on membrane surfaces.

Förster resonance energy transfer-based sensor of steric pressure on membrane surfaces

ACS Spring Meeting 2021

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