Conformational properties of the A-state of cytochrome c studied by hydrogen/deuterium exchange and electrospray mass spectrometry.

Hydrogen/deuterium (H/D) exchange studies that were monitored by liquid chromatography-electrospray ionization mass spectrometry (LC-ESIMS) were used to obtain a structural description of the compact acid-denatured state of ferricytochrome c (A-state). Due to the very different solvent conditions necessary to generate the nonnative states, it was essential that after deuterium labeling the nonnative states were refolded to the native state to insure high reproducibility during sample preparation and LC-ESIMS analysis. Approximately 30% lower deuterium was found incorporated in the A-state compared to the acid-denatured (UA) state. The analysis of the width of the mass peak suggests that the distribution of conformers sampled in the A-state was relatively narrow and that the compactness of the A-state was much closer to that of the native state than to the acid-denatured state. The LC-ESIMS study of partially deuterium-labeled peptic fragments derived from the A-state conformer generated under H/D quenching conditions were interpreted in terms of a significant loss of structural integrity within amino acid region 22-46.