Spectral properties of L-ANAP-ME and Co²⁺-NTA make them an ideal FRET pair for measuring small distances on the order of 10–20 Å. (A) The emission spectrum from L-ANAP-ME conjugated to the bead (black) overlaid on the absorption spectra of Co²⁺-NTA (red), Cu²⁺-NTA (blue), and Ni²⁺-NTA (magenta). (B) Solution quenching of L-ANAP-ME by Co²⁺-NTA. The fluorescence intensities at 490 nm were measured in various concentrations of Co²⁺-NTA in stabilization buffer in a fluorometer. All curves were normalized to their maximum fluorescence intensity after inner filter effect correction and background subtraction. Smooth curve is a fit with the Hill equation with the following parameters: K_1/2 = 25 mM and Hill coefficient = −1. (C) Concentration dependence of quenching of L-ANAP-ME in solution by Co²⁺-NTA (points) fitted with the Hill equation (black curve). (D) Fluorescence lifetime measurements of L-ANAP-ME in the absence (red circles) and presence (blue circles) of 20 mM Co²⁺-NTA. Best fits to the data with a single exponential give time constants of 1.94 and 1.07 ns without and with Co²⁺-NTA, respectively. AU, arbitrary units. (E) Energy transfer efficiencies estimated with steady-state and lifetime fluorescence measurements. The mean for each group is shown by the black line. n.s., no statistically significant difference between the groups (P > 0.05).