Fig. 1.

The significant heterogeneity detected in the FRET efficiency data obtained by the line confocal method of the sm-FRET spectroscopy. The unfolded state of the B domain of protein A, labeled by Alexa488 and ATTO633 to cysteines introduced at 5th and 55th residues near the N and C termini, in the presence of 4 M guanidinium was observed. a presents the FRET efficiency plot for the raw data with the time resolution of 120 μs. b presents the FRET efficiency plots after the moving time average of 2 ms. In a and b, pink bars represent the actual data and the dotted lines represent the theoretical distribution caused by photon shot noise estimated based on the averages of fluorescence intensity, FRET efficiency, and background photons. c presents the examples of the traces observed in the presence of 4 M guanidinium. The dashed lines represent the noise width calculated based on the numbers of fluorescence and background photons for each trace. All data were taken from Oikawa et al. (2015)