| The human membrane proteome is a fundamental cellular "compartment" with thousands of protein components. It is well understood that those proteins rarely function alone and most of them take part in multiple protein-protein interaction complexes. However, probing the cell surface is technically difficult, especially for in vivo experimentation in physiologically relevant conditions. For this purpose a new generation of methods and tools is needed. In this study we developed a novel biosensor for reporting surface protein proximity that exploits the increased avidity effect between a bivalent reporter and two interacting tags. For proof-of-principle a heterobivalent molecule was used with a biotin moiety tethered via a PEG2000 linker to a DIR fluorogenic reporter (biotin-PEG2000-DIR). We designed two fusion proteins and expressed them on the surface of HEK 293 cells: scAvd::FRB, demonstrating high-affinity towards biotin, and HL1::FKBP with low-affinity FAP for DIR. Addition of rapamycin, which promotes FKBP:FRB dimerization, let to an increase in DIR fluorescence due to the proximity of the scAvd and HL1 domains. In a second set of experiments we expanded the biosensor functionality to report on intercellular protein proximity. The increased avidity effect remains true regardless whether the corresponding binding tags are on the same lipid bilayer or separated in different ones. We expressed scAvd and HL1 as transmembrane fusions in two separate HEK 293 populations (Cell A and Cell B accordingly). When biotin-PEG2000-DIR was introduced, the high affinity between the biotin and scAvd drove the ligand to coat the surface of Cell A. At this stage the fluorogenic dye held by the chemical linker was only able to "scan" the surrounding protein neighborhood. When Cell A and Cell B migrated towards one another, so that the surface of Cell B was within reach of the PEG linker, the DIR fluorogen was able to find the HL1-FAP and quickly reported a dramatic increase in fluorescence. This only occurred at the point of contact between the membranes. Additionally, when the membranes moved apart, contact was disrupted and the fluorescent biosensor rapidly turned-off. |
