Tmfret to Study Short-Range Interactions at the Membrane

Abstract

Fluorescence resonance energy transfer (FRET) is a technique used to determine the distance between two fluorophores. However, the precision of the distance measurement is limited by the size of the fluorophores and their linkers. In addition, distances over which traditional FRET pairs are sensitive is on the order of the size of many proteins. Thus, short-range interactions have been difficult to study with FRET.Transition Metal Ion FRET (tmFRET) is a novel technique in which the acceptor fluorophore is replaced with a transition metal divalent cation which acts as a non-fluorescent acceptor. The proximity of a fluorescently labeled protein to the metal ions can be determined by the quenching of the donor fluorophore. The small size of the metal ions coupled with the use of small donor fluorphores, such as bimane, allows precise measurement of short-range interactions on the order of 8-20 A.Here we studied the use of tmFRET for measuring interactions at a cell membrane. As proof-of-principle, we used the membrane-resident dye Octadecyl Rhodamine B Chloride (R18) as a FRET donor and metal-bound stearoyl-NTA as a non-fluorescent quencher. Because stearoyl-NTA and R18 both reside in the membrane and have the same acyl chains, we expect the metal-bound NTA group to be well within the distance range of R18 required for tmFRET. Cells were labeled with R18 via transient introduction into the bath. Perfusion of metal-bound stearoyl-NTA into the bath produced quenching of the R18 fluorescence. We studied the reversibility of this effect by subsequently perfusing the cells with EDTA, which chelates the metal ions. Finally, we determined the concentration dependence of R18 quenching by metal-bound stearoyl-NTA. These experiments lay the groundwork for future studies in which tmFRET will be used to measure the distance of fluorophore-tagged proteins from the membrane.