Many biological functions, involve assembly of proteins. Protein clustering has an important role in signal transduction through the cell membrane. Model systems that simulate receptor aggregation can be used to monitor oligomerization. Techniques based on homo-FRET can reveal the aggregation state of receptor proteins because homo-FRET causes the fluorescence anisotropy of the system to decrease when the number of identical fluorophores within energy transfer distance increases. Theories that describe these systems apply an assumption of equal fluorescence efficiency for all sites [1, 2], that means emission intensity of fluorophores do not change when in close proximity with each other in a cluster. However, most fluorophores in close proximity show either self-quenching or emission enhancement. Previous theories that were devised to study fractional labelling of proteins were only applicable to cases where the distances between the labels were less than 0.8Ro, while in practice the distances between the dyes are more in protein aggregates. Three different systems were chosen to mimic aggregation of fluorescently labelled receptor proteins and analytical expressions were presented for fully labelled and fractionally labelled clusters and the experimental results from the three systems were analyzed. The systems were: a)stochastically labelled BSA containing up to 24 FITC, b) DNA fused trimeric clusters of fluorescein (stochastically labelled), and c)DNA Holliday junctions labelled with fluorescein (in a range of sizes). The inter-probe distances on the DNA constructs were designed to be up to 1.5 Ro to cover cluster sizes of larger protein clusters. The experimental results showed that: 1) none of the clustered species followed the assumption of equal fluorescence efficiency, 2) by applying the assumption of equal fluorescence efficiency, anisotropies were under-predicted and cluster sizes were under-estimated in systems that show quenching and 3) anisotropy behaviour of a multiply labelled cluster of a particular size depends on the behaviour of the fluorophores and their distance in a cluster. As a result of the theory presented here, the size of larger clusters than currently considered possible can be determined; if they are strongly quenched and their fractional labelling is carefully selected
