S1 SingleMolecule FRET States, Conformational Interchange, and Conformational Selection by Dye Labels in Calmodulin Matthew S. DeVore, Adebayo Braimah, David R. Benson, and Carey K. Johnson Department of Chemistry, University of Kansas, Lawrence, KS 66045 Acceptor photobleaching can be a significant problem for singlemolecule FRET measurements. If the FRET acceptor photobleaches within a burst or time bin, the apparent FRET efficiency is less than the true FRET efficiency for the molecule. Excessive photobleaching would shift the FRET efficiency distributions toward lower efficiencies so that the measured distribution no longer accurately reports the populations of FRET states. Acceptor photobleaching occurs most readily for states with high FRET efficiencies because the acceptor is cycled through the excited state more frequently. Donor photobleaching may also bias the FRET efficiency distribution. When the donor photobleaches, signal from that singlemolecule is lost, because the acceptor is not excited at the excitation wavelength. Figure S1 shows the effect of excitation power on cMEM recoveries for CaM34110AF488 TRC2. For this study, no blurring was incorporated into the cMEM model. The excitation power was increased in steps of 10 µW starting at 15 µW average power. As the power increased, the population of high FRET efficiencies decreased, while the population of low FRET efficiencies increased. It was determined that an average excitation power of 25 µW was appropriate to minimize photo bleaching affects in the data. . CaM34,110AF488,TRC2 burst power study. Maximum entropy recoveries of P(E app ) in 300 µs time bins.