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 liqui d chromatography-electrospray ionization mass spectrometry (LC-ESIMS) were used to obtain a structural description of the compact acid-denat ured state of ferricytochrome c (A-state). Due to the very different s olvent conditions necessary to generate the nonnative states, it was e ssential that after deuterium labeling the nonnative states were refol ded to the native state to insure high reproducibility during sample p reparation and LC-ESIMS analysis. Approximately 30% lower deuterium wa s found incorporated in the A-state compared to the acid-denatured (U- A) state. The analysis of the width of the mass peak suggests that the distribution of conformers sampled in the A-state was relatively narr ow 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-sta te conformer generated under H/D quenching conditions were interpreted in terms of a significant loss of structural integrity within amino a cid region 22-46. (C) 1997 Academic Press.