Next, the resulting isolated spectrum was normalized pairwized to the danysl-PE spectrum generated by direct 340 nm excitation to obtain Ratio A. To correct for direct excitation of danysl-PE by 280 nm light, Ratio A 0 was calculated from controls containing only danysl-PE-labeled nanodiscs, which was then subtracted from Ratio A to yield a value directly proportional to FRET efficiency (Ratio A -Ratio A 0 ). Notably, the wavelength independence of FRET efficiency confirmed accurate donor component subtraction and detector linearity.