Nitroxide Spin-Label Quenching of Fluorophore’s Triplet Stateas a Tool for Studying Diffusion Mediated Reactions in LipidMembranes

In this work, we introduce an approach to study bimolecularinteractions in model lipid bilayers and biologicalmembranes, which exploits the influence of membrane-associatedElectron Spin Resonance (ESR) labels on the fluorescencesignal of likewise membrane-bound fluorophoremarkers. It is shown how one can exploit the high detectionsensitivity of the fluorescence signal without loosing the abilityto follow low-frequency molecular interactions, takingplace on a time scale well beyond that of the fluorescencelifetimes. The approach utilizes triplet state monitoring byFluorescence Correlation Spectroscopy (FCS), whereby thefluctuations in a strong fluorescence signal is used to characterizetransition rates to and from the lowest triplet stateof the fluorophores, which take place on a time scale 3 to 6orders of magnitude slower than the fluorescence lifetimesof the fluorophores. FCS measurements were performed onthe dye Lissamine Rhodamine B (LRB) in aqueous solutionsand bound to a lipid in a liposome, and in the presence of differentlocal concentrations of the ESR label TEMPO. Bothin the aqueous solution and in the lipid membrane measurements,the measured relative changes in the singlet-triplettransitions rates were found to well reflect the collisionalfrequencies between the LRB and TEMPO molecules. Theproposed approach, allowing low-frequency interactionsto be monitored with a bright fluorescence signal offers abroad applicability, both in terms of read-out means, typesof molecular interactions that can be followed, and in whatenvironment these interactions can be measured. From thispoint of view, it can prove useful for a broad category ofmolecular interaction studies.QC2010061