Structural characterization of liposome-embedded ncTom40 by H/D exchange coupled to solution-state NMR.
(a) Enlarged spectral regions of [1H, 15N]-HSQC spectra at increasing back-exchange times. To reduce signal overlap, ncTom40 was selectively 15N-labeled at ALA, HIS, ILE, MET, THR. Time points indicate the time after start of the first HSQC. The dissolution buffer contained 75% D2O. Sequence-specific resonance assignments are indicated. (b) Sequence-specific signal intensities in the first HSQC after dissolution in 100% D2O buffer. (c) NMR signal intensity change of residues in panel (a) during back-exchange in 75% D2O. Intensity values were normalized on the basis of the noise level in the spectra. Error bars are based on signal-to-noise. (d) Protonation ratios for residues of Tom40 at the beginning of back exchange. (e) Protonation ratios shown in (d) were mapped onto the topology model of ncTom40, which was predicted on the basis of its homology to hVDAC1. Residues predicted to be in a β-strand or α-helix are boxed. Green-shaded (red-shaded) residues have protonation ratios larger (lower) than 0.3. Residues shown in white were not analyzed due to signal overlap, low signal-to-noise or missing resonance assignment.
