Figure 3.

EPR spectra of R1 incorporated into various locations of CYP3A4 as a function of pressure. The data shown were obtained with no ligand added for all positions of the label except for C85, in which case the spectra recorded in the presence of TST (C85+TST) are also shown for comparison. The main panels show the low-field regions of the spectra, and the complete spectra are shown in the insets. The dashed box in the inset of the top leftmost panel indicates the zoomed-in region in the main panels. EPR spectra are shown for the pressures of 30 (solid line), 1000 (long dash), 2000 (medium dash), and 3000 bar (short dash). Outlined areas in the plots for C85-R1 and C409-R1 indicate the portions of the spectra that display intensity corresponding to mobile (m) and immobile (i) motional states of the nitroxide. In response to an increase in pressure, the spectra of residues C85-R1 and C409-R1 exhibit a shift in population toward the component corresponding to an immobilized state. Subtle changes of the same kind are also detectable for residues C166-R1 and C343-R1.