Theory and application of fluorescence homotransfer to melittin oligomerization.
Fluorescence homotransfer (electronic energy transfer between identical fluorophores) has the potential to quantitate the number of subunits in membrane protein oligomers. Homotransfer strongly depolarizes fluorescence emission as a result of intermolecular excitation energy exchange between an initially excited, oriented molecule and a randomly oriented neighbor. We have theoretically treated fluorescein labeled subunits in an oligomer as a cluster of molecules that can exchange excitation energy back and forth among the subunits within that group. We find that the larger the number of subunits, the more depolarized is the emission. The general equations to calculate the expected anisotropy for complexes composed of varying numbers of labeled subunits are presented. Self-quenching of fluorophores, orientation, and changes in lifetime are also discussed and/or considered. To test this theory, we have specifically labeled melittin on its N-terminal with fluorescein and monitored its monomer to tetramer equilibrium both in solution and in lipid bilayers. The calculated anisotropies are close to the experimental values when non-fluorescent fluorescein dimers are taken into account. Our results show that homotransfer may be a promising method to study membrane-protein oligomerization.
Runnels
L W
LW
Department of Physiology and Biophysics, State University of Stony Brook, New York 11794-8661, USA.
Scarlata
S F
SF
D016650:Fluorescein-5-isothiocyanate; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D008433:Mathematics; D008555:Melitten / Q000737:chemistry*; D008961:Models, Structural; D008962:Models, Theoretical; D017433:Protein Structure, Secondary; D012965:Sodium Chloride; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D008555:Melitten; D012965:Sodium Chloride; D016650:Fluorescein-5-isothiocyanate