Thermal Unfolding of Unsolvated Cytochrome c:  Experiment and Molecular Dynamics Simulations

Abstract

The thermal unfolding of unsolvated cytochrome c has been examined in the gas phase using ion-mobility measurements. Measurements were performed for the +5, +6, and +7 protonated charge states from around room temperature up to 573 K. The (M + 5H)5+ charge state remains folded at 573 K while (M + 6H)6+ and (M + 7H)7+ go through a series of unfolding transitions as the temperature is raised. Molecular dynamics simulations were performed using the CHARMM force field. The simulations are in qualitative agreement with the experimental results: the +7 charge states unfold as the temperature is raised and the +5 charge states remain compact. Addition of two protons to the +5 charge state flattens the energy landscape so that the folded and unfolded conformations have similar energies. Entropy presumably drives the unfolding of the +7 charge state as the temperature is raised.

Temperature-dependent H/D exchange of compact and elongated cytochrome c ions in the gas phase

“…The cross section distributions of the ϩ5 and ϩ9 states vary only slightly with temperature, indicating that there are no large changes in overall shapes of these ions . These results indicate that a compact conformer is most stable for the ϩ5 state (as observed previously) [28,36] and an elongated form is most stable for the ϩ9 state. Although we do not present H/D exchange studies for the ϩ6 to ϩ8 states here, it is noteworthy that experimental results show that thermally-induced structural transitions, similar to those reported previously for the ϩ7 state [30e, 36], are observed over this temperature range.…”

“…At ϳ300 K, the reproducibility of the measured cross sections is excellent; different measurements agree to within 1% of one another (relative uncertainty) and within 2% of values reported previously [28a]. Cross sections determined at high temperatures for the ϩ5 and ϩ7 states are also in good agreement (within a few percent) with values measured previously [36].…”

“…The present work is also closely related to temperature-dependent cross section measurements for the ϩ5 to ϩ7 states carried out by Jarrold and coworkers [36,37]. They find that the ϩ5 state is very stable, retaining a compact conformation even at extreme temperatures (ϳ570 K).…”

“…§ We use the compact NMR structure (the native solution state) as the initial structure for the ϩ5 charge state [36]. The initial structure used for the ϩ9 ion was generated by retaining the ␣-helical regions found in solution as well as forming several short helical sections from regions of the protein primary sequence containing a higher percentage of residues with a greater propensity to form helices.…”

“…As discussed previously [36,39], it is impossible to calculate the total ion energy for all possible charge site assignments for each structure [20a, 36]. Here, we assume formal charges of ϩ3 on the heme iron atom and -1 on each heme carboxylate group [12,40] Added protons from the electrospray process are confined to basic Arg, His, or Lys residues.…”