THEORY AND APPLICATION OF FLUORESCENCE HOMOTRANSFER TO MELITTIN OLIGOMERIZATION

Fluorescence homotransfer (electronic energy transfer between identica l fluorophores) has the potential to quantitate the number of subunits in membrane protein oligomers. Homotransfer strongly depolarizes fluo rescence emission as a result of intermolecular excitation energy exch ange between an initially excited, oriented molecule and a randomly or iented neighbor. We have theoretically treated fluorescein labeled sub units in an oligomer as a cluster of molecules that can exchange excit ation energy back and forth among the subunits within that group. We f ind that the larger the number of subunits, the more depolarized is th e emission. The general equations to calculate the expected anisotropy for complexes composed of varying numbers of labeled subunits are pre sented. Self-quenching of fluorophores, orientation, and changes in li fetime are also discussed and/or considered. To test this theory, we h ave specifically labeled melittin on its N-terminal with fluorescein a nd monitored its monomer to tetramer equilibrium both in solution and in lipid bilayers. The calculated anisotropies are close to the experi mental values when non-fluorescent fluorescein dimers are taken into a ccount. Our results show that homotransfer may be a promising method t o study membrane-protein oligomerization.