Fluorophore labeling and stopped-flow experiments

WT and mutant AFF switch constructs were cloned, expressed, purified, and labeled with BODIPY-FL as described previously¹⁰. The N → N′ and N′ → N conformational changes shorten and lengthen the distance between the BODIPY groups, causing FRET-induced self-quenching to increase and decrease, respectively. The rate constants k_N→N′ and k_N′→N were measured by adding Ca²⁺, i.e., mixing 1 mM CaCl₂ with the WT E65Q and E65′Q constructs, respectively, and monitoring the change in fluorescence intensity by stopped-flow fluorescence. Proteins were desalted into 2.5 M GdnHCl, 20 mM sodium phosphate (pH 7.0), 0.1 mM EDTA using 10DG columns (Bio-Rad), at which point a 3–5 molar excess of the BODIPY-FL maleimide fluorophore (Molecular Probes) was immediately added. The labeling reaction was allowed to proceed in the dark for 2 h at 25 °C. GdnHCl and unreacted BODIPY-FL were removed by passing the sample through a 10DG column equilibrated in 20 mM Tris (pH 7.5), 0.15 M NaCl, 0.2 mM EDTA, and 0.2 % TWEEN-20. BODIPY-FL concentration was measured by absorbance (ε₅₀₃ = 80,000 M⁻¹ cm⁻¹).

Stopped-flow fluorescence data were recorded at 20 °C using Bio-Logic SFM-4000 rapid mixing device and Bio-Logic MOS-200 spectrometer. Excitation was at 487 nm and a 497 nm high-pass filter was used on detection. The fluorescence change was monitored after mixing one volume of BODIPY-FL labeled protein (10 μM) with one volume of 2.1 mM CaCl₂ in 50 mM Tris (pH 7.5), 0.1 % TWEEN-20. Data were analyzed using the Bio-Kine software package (Bio-Logic).

Full details of gene construction, protein expression, and protein purification are provided in Supplementary Methods. Primer sequences are provided in Supplementary Table ³.