Conformational\nDynamics of Hemoglobin in Solution\nand the Gas Phase Elucidated by Mass Spectrometry

Solution and gas-phase measurements can provide valuable\ninsights\ninto biomolecular conformational dynamics. By comparing the data from\nsuch experiments, it is possible to elucidate the nature of the interactions\ngoverning a biomolecule’s stability. Here, we measured human,\nbovine, and porcine hemoglobin stability in solution and the gas phase\n using collision-induced dissociation, collision-induced unfolding,\nsurface-induced dissociation, and temperature-controlled nanoelectrospray\nmass spectrometry. Hemoglobin dimer and tetramer stability in solution\nand gas phases did not correlate, likely due to differences in the\ncomposition of positive and negative amino acids on the surface of\nthese molecules. Specifically, the absence of Lys-116 on the β-subunit\nmakes it easier for the human hemoglobin dimer to dissociate in the\ngas phase. However, the presence of Lys-60 makes the subunit more\nrigid thus it cannot unfold to the same extent as the other hemoglobin.\nHemoglobin tetramers of different origins had similar stability in\nthe gas phase, as there was no difference in the composition of charged\namino acids at the tetramer interface. These results highlight how\ntemperature-controlled mass spectrometry and collision-induced unfolding\ncan elucidate the structural reasons behind differences in the gas-phase\nand solution stability of protein complexes.