Fig. 4.

EPR and ¹⁹F NMR analysis of conformational dynamics of inner loop in response to MAP and temperature. (A) Spectra of Q408C-MTSL (350 μM) before (i) and after addition (ii) of MAP (1.0 mM) (iii) and (iv) are the simulations (red line) of i and ii, respectively. (B) Spectra of K411C-MTSL (350 μM) before (i) and after addition (ii) of MAP (1.0 mM); iii and iv are simulations (red line) of i and ii, respectively. (C) Effect of MAP addition on the spectra of K411C-TFA. The red line is a spectrum of K411C-TFA and shows three resonances at −8.993, −9.106, and −9.193 ppm. The resonance at −9.193 ppm is due to oxidized free label, did not show any ligand-induced changes, and could be removed by extended washing (D). The black trace is a K411C-TFA (250 μM) after addition of saturating amount (500 μM) of MAP. Spectra were acquired at 30°C and referenced to the external standard trifluoroacetate. The data represent 2,048 transients processed with 5 Hz line broadening and a spectral window of 34,617 Hz. (D) Effect of temperature on the ¹⁹F NMR spectra of unliganded K411C-TFA (250 μM). Sample conditions were the same as above.