Figure 4.
Thermodynamic analysis of chemically unmodified and fluorophore- labeled CI2–Cys1′/Cys40. (a) Denaturation of unlabeled CI2– Cys1′/Cys40, followed by the change in Trp-fluorescence emission intensity. (b,c) Denaturation curve of A/D-labeled CI2–Cys1′/Cys40 (b) and D/A-labeled CI2–Cys1′/Cys40 (c). The change in the A-emission after D-excitation (FDA), normalized to A-emission (FA, excitation at 633 nm) is shown. Solid lines in a–c represent fits of the raw data to a two-state unfolding model. (d) Unfolding transitions shown in a–c after normalization to the fraction of folded protein (FN =1 − FU; FU defined by Equation 1b in Materials and Methods). Open circles, unlabeled CI2; open squares, A/D-labeled CI2; open rhombuses, D/A-labeled CI2.