Variable-temperature ESI-IMS-MS analysis of myohemerythrin reveals ligand losses, unfolding, and a non-native disulfide bond.

Variable-temperature electrospray ionization combined with ion mobility spectrometry (IMS) and mass spectrometry (MS) techniques are used to monitor structural transitions of the protein myohemerythrin from peanut worm in aqueous ammonium acetate solutions from similar to 15 to 92 degrees C. At physiological temperatures, myohemerythrin favors a four-helix bundle motif and has a diiron oxo cofactor that binds oxygen. As the solution temperature is increased from similar to 15 to 35 degrees C, some bound oxygen dissociates; at similar to 66 degrees C, the cofactor dissociates to produce populations of both folded and unfolded apoprotein. At higher temperatures (similar to 85 degrees C and above), the IMS-MS spectrum indicates that the folded apoprotein dominates, and provides evidence for stabilization of the structure by formation of a non-native disulfide bond. In total, we find evidence for 18 unique forms of myohemerythrin as well as information about the structures and stabilities of these states. The high-fidelity of IMS-MS techniques provides a means of examining the stabilities of individual components of complex mixtures that are inaccessible by traditional calorimetric and spectroscopic methods.