Characterization of BRET efficiency and spectral contribution of BRET pairs.

Absorbance (λAbs) and emission maxima (λEm), molar extinction coefficient (ε), emission quantum yield (QY), brightness (BR = εQY/1000), taken from references [22], [23], [31], [32], were used for the calculation of BRET parameters. The percentage contribution of aequorin (Aeq) bioluminescence and six BRET pairs into four emission channels was measured in HeLa cells expressing chimeras during Ca2+ responses (see Materials and Methods). Raw counts were corrected for filter bandwidth and transmittance, and also for camera sensitivity (manufacturer's data); the counts in each channel were divided by the sum of the counts in the four channels, and were expressed as a percentage. The average percentage of 15 to 49 cells ± S.D. is shown. Filters (Figure S2A) are named as their center wavelength. The theoretical BRET parameters, J (λ) and R0, are the spectral overlap integral and Förster distance (see Materials and Methods for details).