FIGURE 8.

Exposure of different RTA residues to the membrane interior. A, the tube worm representation of the α-carbon backbone of the RTA crystal structure is shown. Amino acids that are exposed to the membrane at 20 °C are shown in green, whereas amino acids that are exposed to the nonpolar lipid core only at 37 °C are indicated in red. B, the emission intensities of NBD-labeled RTA mutants (450 nm in buffer H) were measured before and after the addition of PCPS liposomes. Emission intensities of parallel samples containing either 22.5 mol% 12NOPC (F_12NO) or 22.5 mol% of PC (F₀) were compared at 20 °C (gray bars) or at 37 °C (black bars), respectively. The averages of at least three independent experiments are shown, and the error bars indicate the S.D. of the experiments. Sequence numbers of NBD-labeled amino acids are shown on the x axis. ^*, p = 0.0004; ^**, p = 0.04; ^***, p = 0.07 when compared with the corresponding quenching efficiency at 20 °C (Student's t test). C and D, the ratio of RTA mutant (450 nm in buffer H) NBD emission intensity (C) and the change in λ_{em max} (D) are shown before (F₀) and after (F_KRM) binding to KRMs (20 eq), either at 20 °C (gray bars) or at 37 °C (black bars). The average of at least three independent experiments is shown, and the error bars indicate the S.D. of the experiments. A, ^*, p < 0.00004; ^**, p < 0.0002. B, ^*, p < 0.00001; ^**, p < 0.001 when the measurements at 20 °C were compared with those at 37 °C (Student's t test).