Skip to main content
. Author manuscript; available in PMC: 2013 Aug 1.
Published in final edited form as: J Am Chem Soc. 2012 Jul 19;134(30):12565–12577. doi: 10.1021/ja302528z

Figure 3.

Figure 3

Comparison of relative FRET-efficiencies for the denatured subpopulation measured by equilibrium smFRET (circles) and the asymptotic FRET-efficiency of the time-resolvable microsecond kinetic phase measured by ultrafast laminar-flow mixing (triangles). A comparison of relative FRET efficiencies was necessary to account for minor differences in detection efficiencies between the microscopic setups used for the smFRET and ensemble mixing experiments and the presence of donor-only species in the ensemble mixing experiment that were digitally removed in the smFRET experiments. For the smFRET experiments, raw FRET efficiencies of the denatured subpopulation at a particular denaturant concentration were normalized to the difference in FRET efficiency between the folded subpopulation at 0 M GuHCl, and the FRET efficiency of the denatured subpopulation at 6 M GuHCl. For the ensemble mixing experiments, raw asymptotic FRET efficiencies for the microsecond phase at a particular denaturant concentration were normalized to the difference in FRET efficiency of the denatured protein at 6 M (unfolded baseline in Fig. 3a, main text) and the folded protein at 0 M (folded baseline in Fig. 3a, main text). Note that some asymptotic FRET values are not shown: W55F (6 M to 3 M), Y31N (6 M to 1.5 M) and Y31N (6 M to 3 M); these traces were poorly fit by a single-exponential.