Figure 1.

Spectral characteristics of resonance energy transfer probes. The emission spectrum of terbium phosphorescence of the unconjugated terbium chelate in distilled water was collected at a 2-nm bandwidth with the excitation monochromator set at 342 nm. Excitation at 540 nm of a stained rat skeletal muscle cryostat section yielded the tetramethylrhodamine phalloidin-labeled actin fluorescence emission curve. The absorption spectrum of tetramethylrhodamine phalloidin-labeled actin (purified rabbit skeletal muscle actin) was measured on a diode array spectrometer with a bandwidth of 1 nm in 2 mM MgCl₂/0.2 mM CaCl₂/0.2 mM ATP/0.5 mM 2-mercaptoethanol/2 mM Tris (pH 7.0). Unlabeled actin did not absorb in the part of the spectrum shown. (Inset) Dependence of efficiency of energy transfer (E) on separation distance (R) between a donor and acceptor probe pair. Using the inset equation, the theoretical relationship between E and R was calculated for critical transfer distance (R_o) of 59 Å. The critical transfer distance was calculated from the overlap between the donor’s emission spectrum and the acceptor’s absorbance spectrum. (See text for calculations.)