A novel FRET-based DNA nanosensor to quantify clustering forces of membrane proteins.

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Membrane proteins play a crucial role in various biological phenomena such as endocytosis and signal transduction. These phenomena are heavily dependent on the clustering of membrane proteins. While the propensity of proteins to cluster on the cell membrane has been well established, the physical mechanisms that govern this behavior are far from clear. Furthermore, the tools to be able to measure the forces responsible for the clustering of membrane proteins have not been developed yet. To bridge this gap, we have combined elements from biophysics, cellular and chemical biology, and biochemistry, to design a FRET-based DNA nanosensor. Our results demonstrate that an increase in the stiffness of the sensor increases the distance between the two dyes, which is obtained via FRET efficiency measurements. The sensor, once calibrated for stiffness, will be combined with this distance measurement to obtain the force that drives the interaction between pairs of proteins. As a proof of concept, the sensor design and measurements are being done using the B-subunit of the Shiga Toxin (STxB). Following measurements on STxB proteins, this novel method shall then be applied to understand the clustering behaviour of other membrane proteins.