Time-resolved electrospray ionization (ESI) mass spectrometry (MS) is a new technique for studying the kinetics of protein folding reactions. It can monitor both changes in the protein conformation and the loss or binding of protein ligands as a function of time. Time-resolved ESI MS was previously used to monitor the acid-induced unfolding of holomyoglobin (hMb). The native form of this protein is characterized by a tightly folded conformation and a heme group that is noncovalently attached to the protein. Acid-induced denaturation induces substantial unfolding of the polypeptide chain and disruption of the heme-protein interactions. In this work, time-resolved ESI MS is used to study the reverse reaction, i.e., reconstitution of acid-denatured hMb. To examine the mechanism and the kinetics of this reaction, a continuous-flow setup with two sequential mixing steps was developed. The data presented in this work show that reconstitution involves the formation of various short-lived intermediates such as tightly folded myoglobin without a heme group and several nativelike forms of the protein that are bound to more than one heme. The occurrence of these transient states is most likely due to the rapid aggregation of free heme in solution.

No abstract

Near pH 2.0, lysozyme in water is in its native conformation, and in water/methanol (2/8) it adopts a helical denatured conformation (Kamatari et al. Protein Sci. 1998, 7, 681-688). Hydrogen/deuterium (H/D) exchange of lysozyme in solution confirms that it is partially unfolded at pH 2.0 in water/methanol (v/v = 2/8). With electrospray ionization (ESI) mass spectrometry (MS), lysozyme in water produces ions with charges +7 to +12, with the greatest intensity at +10, whereas lysozyme in water/methanol (2/8) produces ions with charges +6 to +12 with the greatest intensity at +7. Thus, lysozyme is an exception to the rule that a protein denatured in solution forms higher charge states than the same protein in its folded native conformations in solution. Because the same charge states are produced from these two solution conformations, a direct comparison of the properties of the gas-phase ions produced from two very different solution conformations is possible. The conformations of lysozyme ions in the gas phase were studied using cross section measurements and gas-phase H/D exchange. Similar cross sections and H/D exchange levels were observed for same-charge states of lysozyme ions formed from the native and helical denatured conformations in solution. Cross sections show that the ions have compact structures. Thus, disulfide-intact gaseous lysozyme ions generated from the denatured state in water/methanol (2/8) refold into compact structures in the gas phase on a time scale of milliseconds or less.

Gas phase holomyoglobin (hMb) ions in charge states +7 to +21 were formed by electrospray ionization in combination with a continuous-flow mixing apparatus. Collision cross section measurements show that the highly charged ions are somewhat unfolded in comparison to low charge states but still retain a considerable degree of folding. A new collision model is presented which calculates the relative energies transferred to complexes in tandem mass spectrometry. Tandem mass spectrometry and ion trapping experiments both show that the energies required to dissociate heme from the highly charged heme-protein complexes in the gas phase are similar to those of low charge states, previously shown in literature ion cyclotron resonance experiments to be 0.7-1.0 eV. These energies are comparable to those of the heme binding energy in solution. The results suggest that even for the highly charged hMb ions which have unfolded somewhat, the heme-protein interactions remain relatively unperturbed.