Transition Metal Ion FRET Measurements in Plasma Membrane Sheets Prepared by Deroofing Cultured Mammalian Cells

Abstract

Since the discovery of Forster resonance energy transfer (FRET) in the 1940s, its implementation to measure inter- and intramolecular distances has reached wide-spread use in the biological sciences. Because the technique requires labeling of a donor and acceptor site with two different spectroscopically active molecules, a prominent difficulty with biological samples has been how to label with specificity and, therefore, unambigouisly detect and assign the signal contribution from each site. Here we show that transition metal ion FRET (tmFRET) experiments using plasma membrane sheets prepared from deroofed cells can be used as an orthogonal labeling scheme with an unambiguous assignment of the FRET pair signal. As a FRET donor, we used octadecyl rhodamine B (R18), which incorporated into the plasma membrane of deroofed cultured cells. To measure distances on the length scale relevant to membrane proteins, we used the transition metal ion Co2+ as an acceptor, which, together with R18, gives a R0 value of 14.9 A. Although the quenching by Co2+ of rhodamine B in solution is of low affinity (>10 mM), 100 μM Co2+ caused appreciable quenching of R18 incorporated into plasma membrane sheets. By first introducing a synthetic fatty acid with a metal-chelating head group, appreciable quenching was observed with less than 1 μM Co2+. Comparison of tmFRET experimental measurements to expected theoretical values convincingly demonstrate that these experiments provide information about the distance of closest approach between donor and acceptor (Fung and Stryer, 1978). These experiments provide proof-of-priniciple for measurements of biologically relevant distances in native membranes. We anticipate that FRET experiments on plasma membrane sheets will offer a unique opportunity to explore protein-lipid interactions and discuss several possible future directions for this technique.