Figure 17.9.5.

Simulations of FRET for assembled versus unassembled oligomers. (A) Idealized configurations of an oligomeric channel in the membrane in an assembled and disassembled configuration. The positions of a putative antigen present as three copies in the channel are indicatedwithablack dot.(B) Surface view of a 70 × 70–nm section of membrane containing antigens (black dots) at 2000 copies per mm² in assembled and disassembled configurations as in (A). The two dimensional projection of the positions of donor (green) and acceptor (red) dyes on antibodies bound to these antigens is also indicated. An antibody was assigned a 20% probability of containing donor dyes. (C) Monte Carlo simulations were used to calculate the FRET efficiencies in a 0.25 μm² section of membrane containing donor and acceptor labeled antibodies (at a donor/acceptor ratio of 1:4, each containing 4 dye molecules per IgG) bound to antigens (with 100% occupancy) at densities of 1000 to 5000 copies per μm². The antigens were either randomly distributed or assembled into clusters of three (around a 10-nm circle) that were randomly distributed in the membrane surface (closed circles). The simulation was repeated ten times for each condition, with each point representing the FRET from a single simulation. Note that FRET in the nonclustered configuration displays a clear density-dependence that is not seen in the clustered configuration. (C-E) FRET as a function of the degree of occupancy of the antigen by antibody, and proportion of antibodies that carry donor dyes were simulated as in (C). Randomly distributed antigens (open circles) were compared to clustered antigens (closed circles). The mean ± SD of ten simulations is plotted for each condition. When not being specifically varied, antibodies contained four dyes each, 20% of antibodies were donors, antigen occupancy was 100%, and antigen density was 2000 copies per μm².