(a) The autocorrelation function (ACF) of W144/C199-Atto moPrP is shown in red. The ACF of free Atto dye, acquired under identical conditions, is shown in blue. The ACF of the control Trp-less variant, C199-Atto moPrP, is shown in green. The fits to appropriate models are shown in black for each ACF. (b) The ACF of W171/C225-Atto moPrP, free Atto dye and the Trp-less control protein C225-Atto moPrP are shown in red, blue and green, respectively. The fits to appropriate models are shown in black for each ACF. The values of τd for free Atto dye, W144/C199-Atto moPrP and W171/C225-Atto moPrP are 66 ± 5, 304 ± 22 and 316 ± 30 µs. (c) Atto fluorescence emission spectra of W144/C199-Atto moPrP (red) and the corresponding Trp-less control protein C199-Atto moPrP (green) at pH 4, are shown. (d) Atto 655 fluorescence emission spectra of W171/C225-Atto moPrP (red) and corresponding Trp-less control protein C225-Atto moPrP (green) at pH 4, are shown. In panels c and d, the spectra of the native proteins are shown as continuous lines; the insets show the spectra of the unfolded proteins (in 6 M Urea) as dashed lines (inset). The spectra were normalized to the fluorescence of the Trp-containing protein at 690 nm.