When One Plus One Does Not Equal Two: Fluorescence Anisotropy in Aggregates and Multiply Labeled Proteins

The behavior of fluorescence anisotropy and polarization in systems with multiple dyes is well known. Homo-FRET and its consequent energy migration cause the fluorescence anisotropy to decrease as the number of like fluorophores within energy transfer distance increases. This behavior is well understood when all subunits within a cluster are saturated with fluorophores. However, incomplete labeling as might occur from a mixture of endogenous and labeled monomer units, incomplete saturation of binding sites, or photobleaching produces stochastic mixtures. Models in widespread and longstanding use that describe these mixtures apply an assumption of equal fluorescence efficiency for all sites first stated by Weber and Daniel in 1966. The assumption states that fluorophores have the same brightness when free in solution as they do in close proximity to each other in a cluster. The assumption simplifies descriptions of anisotropy trends as the fractional labeling of the cluster changes. However, fluorophores in close proximity often exhibit nonadditivity due to such things as self-quenching behavior or exciplex formation. Therefore, the anisotropy of stochastic mixtures of fluorophore clusters of a particular size will depend on the behavior of those fluorophores in clusters. We present analytical expressions for fractionally labeled clusters exhibiting a range of behaviors, and experimental results from two systems: an assembled tetrameric cluster of fluorescent proteins and stochastically labeled bovine serum albumin containing up to 24 fluorophores. The experimental results indicate that clustered species do not follow the assumption of equal fluorescence efficiency in the systems studied with clustered fluorophores showing reduced fluorescence intensity. Application of the assumption of equal fluorescence efficiency will underpredict anisotropy and consequently underestimate cluster size in these two cases. The theoretical results indicate that careful selection of the fractional labeling in strongly quenched systems will enhance opportunities to determine cluster sizes, making accessible larger clusters than are currently considered possible.