Characterization of Intramolecular Interactions of Cytochrome c Using Hydrogen-Deuterium Exchange-Trapped Ion Mobility Spectrometry-Mass Spectrometry and Molecular Dynamics

Abstract

Globular proteins, such as cytochrome c (cyt c), display an organized native conformation, maintained by a hydrogen bond interaction network. In the present work, the structural interrogation of kinetically trapped intermediates of cyt c was performed by correlating the ion-neutral collision cross section (CCS) and charge state with the starting solution conditions and time after desolvation using collision induced activation (CIA), time-resolved hydrogen/deuterium back exchange (HDX) and trapped ion mobility spectrometry-mass spectrometry (TIMS-MS). The high ion mobility resolving power of the TIMS analyzer allowed the identification of new ion mobility bands, yielding a total of 63 mobility bands over the +6 to +21 charge states and 20 mobility bands over the -5 to -10 charge states. Mobility selected HDX rates showed that for the same charge state, conformers with larger CCS present faster HDX rates in both positive and negative ion mode, suggesting that the charge sites and neighboring exchange sites on the accessible surface area define the exchange rate regardless of the charge state. Complementary molecular dynamic simulations permitted the generation of candidate structures and a mechanistic model of the folding transitions from native (N) to molten globule (MG) to kinetic intermediates (U) pathways. Our results suggest that cyt c major structural unfolding is associated with the distancing of the N- and C-terminal helices and subsequent solvent exposure of the hydrophobic, heme-containing cavity.

Figure 1

Left panel typical mass spectra of cyt c as a function of the starting solvent conditions. Right panel: overall CCS profiles (black lines) obtained by summation of the intensity-normalized IMS resolved data (color lines). The results obtained in the positive and negative ion mode are represented on the top and bottom part, respectively.

Figure 2

Left panel IMS spectra of the +6 to +11 charges states of cyt c as a function of the organic content (e.g., % methanol). Right panel: IMS spectra of the +6 to +11 charge states of cyt c as a function of the activation energy (e.g., deflector voltage).

Figure 3

Bottom: IMS profiles obtained with CIA (blue line), and without CIA (green line). Top: High resolution IMS profiles for the +12 to +21 charge states of cyt c.

Figure 4

HDX back exchange as a function of the incubation time (e.g., 3 and 48 h) and the time after desolvation (e.g., 0, 100, and 500 ms), in terms of charge state and CCS in the positive ion mode. Clustering of the number of kinetically trapped intermediates and the initial levels of exchange allows the identification of four groups with different levels of exchange protection (color rectangles and data points).

Figure 5

Candidate structures proposed for the kinetic intermediates IMS bands of cyt c. Conformations G and H for the +7 charge state (dark red background); and C, D, and E (dark green background), for the +8, and +9 charge states, were obtained after CIA.